# A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection

Xue Liu, Laurye van Maele, Laura Matarazzo, Daphnée Soulard, Vinicius

Alves Duarte da Silva, Vincent de Bakker, Julien Dénéréaz, Florian Bock,

Michael Taschner, Jinzhao Ou, et al.

► To cite this version:

Xue Liu, Laurye van Maele, Laura Matarazzo, Daphnée Soulard, Vinicius Alves Duarte da Silva, et al. A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection. Cell Host & Microbe, 2024, 32 (3), pp.304-314.e8. 10.1016/j.chom.2024.02.002. inserm-04605118

HAL Id: inserm-04605118

https://inserm.hal.science/inserm-04605118v1

Submitted on 14 May 2025

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# Cell Host & Microbe

## A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection

### Graphical abstract

CRISPRi screening

<figure><img src="image_2.png" /></figure>

Pneumococcal CRISPRi library

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

sampling of lung bacteria

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

LafB as the target

<figure><img src="image_7.png" /></figure>

The virulence factor

<figure><img src="image_8.png" /></figure>

Membrane-associated intracellular glycosyltransferase

<figure><img src="image_9.png" /></figure>

Immunogenic in human

Mucosal vaccine protection

LafB+Adjuvant

<figure><img src="image_10.png" /></figure>

Intranasal vaccination

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

Lung-resident memory Th17 lymphocytes

<figure><img src="image_13.png" /></figure>

IL-17

<figure><img src="image_14.png" /></figure>

Serotype-independent protection

### Authors

Xue Liu, Laurye Van Maele, Laura Matarazzo, ..., Victor Nizet, Jean-Claude Sirard, Jan-Willem Veening

### Correspondence

jean-claude.sirard@inserm.fr (J.-C.S.), jan-willem.veening@unil.ch (J.-W.V.)

### In brief

Liu et al. identify *lafB* as crucial for *Streptococcus pneumoniae* replication *in vivo* using CRISPRi-seq. Intranasal vaccination with flagellin-adjuvanted LafB induces lung Th7 lymphocytes that protect against superinfections with various pneumococcal serotypes in mice. Healthy individuals can elicit LafB-specific immune responses, suggesting that LafB is a universal, capsule-independent pneumococcal antigen.

### Highlights

* CRISPRi-seq in pneumococcal superinfection identified *lafB* as crucial for virulence

* LafB catalyzes the formation of a glycolipid important for cell wall homeostasis

* Intranasal vaccination with LafB protects against pneumococcal non-vaccine serotypes

* Nasal vaccine-induced protection depends on lung Th17 lymphocytes with TRM features

<figure><img src="image_15.png" /></figure>
Short article

# A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection

Xue Liu,<sup>1,2,6</sup> Laurye Van Maele,<sup>3,6</sup> Laura Matarazzo,<sup>3</sup> Daphnée Soulard,<sup>3</sup> Vinicius Alves Duarte da Silva,<sup>3</sup>

Vincent de Bakker,<sup>2</sup> Julien Dénéréaz,<sup>2</sup> Florian P. Bock,<sup>2</sup> Michael Taschner,<sup>2</sup> Jinzhao Ou,<sup>1</sup> Stephan Gruber,<sup>2</sup> Victor Nizet,<sup>4,5</sup> Jean-Claude Sirard,<sup>3,*</sup> and Jan-Willem Veening<sup>2,4,7,*</sup>

## SUMMARY

Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen *Streptococcus pneumoniae* that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved *lafB* gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against *S. pneumoniae* serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection.

## INTRODUCTION

*Streptococcus pneumoniae* is a leading cause of bacterial pneumonia and a major cause of death and disability in young children and aged or immunocompromised adults. Notoriously, *S. pneumoniae* proves particularly virulent in combination with antecedent influenza A virus infection. Such secondary pneumococcal infections, or superinfections, contribute significantly to excess morbidity and mortality in high-risk groups, as highlighted during the influenza pandemics of 1918, 1957, 1968, and 2009.<sup>1-4</sup>

Currently, pneumococcal vaccines are capsule polysaccharide (CPS)-based, such as Prevenar 13, which is composed of 13 CPSs conjugated to a carrier protein and Pneumovax, the pneumococcal polysaccharide vaccine (PPSV) that contains 23 CPSs.<sup>5</sup> Whereas both vaccines elicit CPS-specific antibodies, the conjugated vaccine induces T cell-dependent immunity, which elicits stronger and long-term antibodies.<sup>6,7</sup> While these

vaccines are successful in reducing the burden of infections caused by 13–23 serotypes, they do not protect against invasive pneumococcal disease caused by non-vaccine serotypes.<sup>8,9</sup> There are more than 100 known serotypes of *S. pneumoniae*,<sup>10</sup> and serotype replacement and appearance of non-typeable clinical isolates reduce the efficacy of CPS-based vaccines.<sup>11,12</sup> To encompass a broader range of serotypes, recent vaccine developments include Prevenar 20, which targets 20 serotypes,<sup>13</sup> and Vaxneurance, covering 15 serotypes.<sup>14</sup> However, even with these advancements, only a subset of the serotypes are covered. Importantly, CPS-based vaccines provide poor protection during pneumococcal superinfection in mice.<sup>15,16</sup> How well CPS-based vaccines work in the context of superinfection is unclear from human vaccine studies.<sup>17</sup> What is clear is that influenza infection decreases pneumococcal clearance and increases lung injury even in PPSV-vaccinated mice.<sup>16</sup> Conversely, pneumococcal colonization may also impede mucosal responses to live attenuated influenza vaccine,

<footnote n="1"><sup>1</sup>Department of Pathogen Biology, Base for International Science and Technology Cooperation, Carson Cancer Stem Cell Vaccines R&D Center, International Cancer Center, Shenzhen University Medical School, Shenzhen 518060, China</footnote>

<footnote n="2"><sup>2</sup>Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, CH-1015 Lausanne, Switzerland</footnote>

<footnote n="3"><sup>3</sup>University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, 59000 Lille, France</footnote>

<footnote n="4"><sup>4</sup>Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA</footnote>

<footnote n="5"><sup>5</sup>Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA</footnote>

<footnote n="6"><sup>6</sup>These authors contributed equally</footnote>

<footnote n="7"><sup>7</sup>Lead contact</footnote>

<footnote>*Correspondence: jean-claude.sirard@inserm.fr (J.-C.S.), jan-willem.veening@unil.ch (J.-W.V.)
https://doi.org/10.1016/j.chom.2024.02.002</footnote>
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 1.** LafB is an essential virulence determinant</figcaption></figure>

(A) Workflow of CRISPRi-seq using injected doxycycline. Mice (n = 5) were inoculated intranasally with the CRISPRi library.
(B) CRISPRi knockdown of the capsule operon (cps2A-N, cps2F-N) and *lafB* results in reduced fitness in mice, compared with *in vitro* (C + Y medium).
(C) Competition index of individual mutants, compared with wild-type (WT) D39V. Strain VL3200 is similar to WT but contains an erythromycin resistance marker at a neutral locus. Each datapoint represents the lung colony forming unit (CFU) count at day 8 of a single mouse infected with flu at day 0 and a ratio 1:1 of mutant and WT strain at day 7. Twelve animals were included in each group.
(D) The *ΔlafB* mutant was attenuated in establishing lung infection (11–12 mice per group). Ectopic expression of *lafB* complemented the phenotype. Kruskal-Wallis testing was used to compare groups.

including reduced production of nasal immunoglobulin A (IgA) and lung IgG in humans.¹⁸

Thus, there is an urgent need for an efficient vaccine that protects against any pneumococcal strain. A promising avenue for a universal, serotype-independent vaccine is in the use of immunogenic proteins as protective antigens.⁵,¹⁹⁻²⁷ So far, efforts have been focused on surface-exposed proteins as these might be directly recognized by opsonizing antibodies. However, surface-exposed proteins typically show significant strain-to-strain sequence variability because of antigenic variation.²⁸⁻³⁰ To uncover universal antigens, an unbiased genome-wide vaccinology approach is required. Previous attempts have used transposon insertion sequencing (Tn-seq) to identify pneumococcal antigens.³¹,³² While successful, these approaches identified non-essential genes encoding variable surface-exposed proteins that suffer from the limitations outlined above. Here, employing CRISPR interference (CRISPRi) that allows the interrogation of essential genes,³³ we searched specifically for conserved genes important for bacterial survival during superinfection. We show that one hit, lipoteichoic acid anchor formation protein B (LafB), a highly conserved membrane-associated protein, is important for cell wall homeostasis and crucial for virulence.

Importantly, recombinant LafB provides broad T helper 17 (Th17) cell-specific protective immunity, paving the way for a universal, capsule-independent pneumococcal vaccine.

## RESULTS

### CRISPRi-seq identifies S. pneumoniae LafB as crucial for virulence during influenza superinfection

Using CRISPRi-seq of a genome-wide sgRNA library in mice fed doxycycline-containing food, we confirmed pneumococcal capsule as an important virulence factor during superinfection.³⁴ To precisely control *in vivo* dCas9 expression, doxycycline levels were optimized following intraperitoneal (i.p.) injection in mice. The *ex vivo* CRISPRi-based luciferase assay found that as little as 4 ng/ml doxycycline repressed luciferase transcription >15-fold (Figures S1A–S1D); i.p. injection of 5 mg/kg doxycycline adequately activated the pneumococcal CRISPRi system in the lung.

Next, mice were infected intranasally (i.n.) with H3N2 influenza virus, followed at day 7 by i.n. infection with the S. pneumoniae CRISPRi library. dCas9 was induced by doxycycline and compared with mock (vehicle) control (Figure 1A). CRISPRi-seq
A

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

B

<figure><img src="image_20.png" /></figure>

C

<figure><img src="image_21.png" /></figure>

D

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

E

<figure><img src="image_24.png" /></figure>

F

<figure><img src="image_25.png" /></figure>

G

<figure><img src="image_26.png" /><figcaption>**Figure 2.** LafB is a membrane-associated galactosyl-glucosyl-diacylglycerol synthase with a pleiotropic role in cell wall homeostasis</figcaption></figure>

(A) LafB is encoded in the same operon with LafA. LafA catalyzes the synthesis of glucosyl-diacylglycerol (Glc-DAG), which provides the anchor for lipoteichoic acids (LTA). LafB catalyzes the addition of galactose onto Glc-DAG synthesizing GalGlc-DAG (Figure S2).

(B) The predicted structure of LafB by RoseTTAFold. Negative and positive electrostatic potentials are colored red and blue, respectively. The two blue arrows point to the active units. No transmembrane domain was identified.

(C) Fluorescence microscopy analysis of GFP-LafB showed a membrane localization.

(legend continued on next page)
<figure><img src="image_27.png" /></figure>

confirmed the capsule operon as critical for pneumococcal viru-
lence; in contrast, the *in vitro* essential gene *metK* was dispens-
able *in vivo*³⁴ (Figure 1B). To pinpoint conserved S. pneumoniae
genes with important virulence functions, we plotted the fitness
values of each clone across *in vitro* and *in vivo* conditions. This
analysis identified sgRNA0370 targeting the gene *spv_0960*
(lafB, a.k.a. *cpoA*), previously deemed as essential in Tn-seq ex-
periments,³⁵ to be significantly underrepresented *in vivo* (Fig-
ure 1B; Table S1).

To validate the CRISPRi-seq screen, *lafB*-deleted and -com-
plemented mutants were constructed (Figures S1E and S1F).
Competition assays were conducted 7 days post influenza infec-
tion with wild-type (WT) *S. pneumoniae* paired with a *lafB* mutant
or a *cps* mutant (avirulent control). *S. pneumoniae* lacking LafB
were outcompeted by WT bacteria in lungs (Figure 1C). These re-
sults were confirmed in single-strain superinfection experiments
as *lafB* mutant bacteria were significantly reduced in bacterial
counts, compared with the WT or *lafB*-complemented strains
(Figure 1D). Invasive disease, assessed by splenic dissemina-
tion, was likewise attenuated in animals infected with the *lafB*
mutant (Figures S1G and S1H).

LafB is an intracellular membrane-associated protein
involved in cell wall homeostasis

LafB (347 amino acids, 40 kDa)³⁶ is conserved among pneumo-
cocci (>96% amino acid identity in all sequenced pneumococci)
(Figures S1J and S1K) and is implicated in the production of galac-
tosyl-glucosyl-diacylglycerol, a glycolipid of unknown function
(Figure 2A).³⁷,³⁸ Incubation of recombinant LafB with α-monoglu-
cosyldiacylglycerol (mGlc-DAG) and UDP-galactose followed
by mass spectrometry, demonstrated the production of UDP
(Figures S2A-S2D), establishing that LafB is a diglucosyl diacylgly-
cerol synthase, as proposed previously.³⁹ Additionally, *lafB*-defi-
cient pneumococci have a slightly reduced susceptibility to peni-
cillins³⁷ but an increased susceptibility to daptomycin and acidic
stress.³⁸,⁴⁰ Prior immunoblot analysis found that LafB co-purifies
with the membrane fraction.³⁷ However, while our structure pre-
diction using RoseTTAFold⁴¹ demonstrates the Rossmann-like
domain of GT-B glycosyltransferases,⁴² transmembrane domains
were not detected (Figure 2B). Overlay of our predicted model of
LafB to a related GT-B glycosyltransferase, *Mycobacterium
tuberculosis* PimA (PDB: 2GEK),⁴³ showed good agreement, albeit
with deviations in the active site cleft (Figure S1M).

To pinpoint LafB cellular localization, we constructed a func-
tional GFP-LafB fusion (Figures S1I and S1L) and performed
fluorescence microscopy. As shown in Figure 2C, GFP-LafB
demonstrates membrane-associated localization. Split comple-
mentation luciferase assays for topology showed that both LafB
termini reside in the cytoplasm (Figure 2D). The intracellular
localization of LafB was further substantiated using a LafB-
specific antibody binding assay (Figures S2F-S2H). These data
support that LafB is an intracellular protein that is associated

via hydrophobic and charge interactions with the cytoplasmic
membrane.

To gain insight into *lafB* mutant virulence attenuation, we per-
formed a synthetic lethal screen by introducing a genome-wide
sgRNA library into the Δ*lafB* mutant background (Figure 2E).
As shown in Figure 2F, the gene encoding the division protein
DivIB<sup>44</sup> becomes essential in a *lafB* mutant, suggesting that
galactosyl-glucosyl-diacylglycerol plays a role for efficient cell
division. The gene *cozE* (a.k.a. *cozEa*) encoding a regulator of
penicillin-binding protein Pbp1A<sup>45</sup> becomes less essential in
the absence of *lafB* (Figure 2F). This genetic interaction may
reflect prior findings that *cozE* mutants have deranged Pbp1A
activity, causing cell lysis.<sup>45</sup> Since *lafB* mutants have reduced
Pbp1A levels,<sup>37</sup> a double *lafB/cozE* knockdown alleviates the
*cozE* single-mutant phenotype. Testing individual knockdowns
of *divIB* and *cozE* validated the screen (Figure 2G).

Given that LafB uses the same lipid anchor as TacL to produce
lipoteichoic acids (LTA),⁴⁶,⁴⁷ we hypothesized that *lafB* deletion
would accumulate the substrate for LTA (Figure 2A), resulting
in an increased quantity of teichoic acids. Therefore, we quanti-
fied the phosphorylcholine levels in the *lafB* mutant using a spe-
cific antibody (TEPC-15) and flow cytometry. Indeed, the *lafB*
mutant displayed a higher amount of phosphorylcholine on its
cell surface than WT (Figures S2I and S2J). This observation
suggests that LafB plays a crucial role in the homeostasis of tei-
choic acids.

Vaccination with LafB induces antigen-specific adaptive immune responses

To establish whether LafB is a protective antigen, we produced
the S. pneumoniae D39V LafB protein in E. coli (Figure S2A). Re-
combinant LafB was formulated with alum as adjuvant for sub-
cutaneous (s.c.) immunization or with the recombinant bacterial
flagellin FliCΔ174-400 as an adjuvant⁴⁸⁻⁵⁰ for i.n. immunization.
Flagellin has emerged as a safe and potent adjuvant by respira-
tory route against a variety of pathogens.⁵¹ Immune responses
specific for LafB were tested in mice on day 28 after a prime-
boost vaccination (Figure 3A). A strong LafB-specific antibody
response (IgG and IgM but no IgA) was observed for s.c.-vacci-
nated animals in serum and broncho-alveolar lavages, respec-
tively (Figures 3B and S3A-S3D). In contrast, LafB-specific anti-
bodies were weakly elicited in mice vaccinated via the i.n. route.
When immune cells from lung, spleen, and mediastinal lymph
nodes (MdLN) were stimulated ex vivo with LafB, cytokines
associated with Th1 (IFN-γ), Th2 (IL-13), and Th17 (IL-17/IL-22)
were produced in response, regardless of the vaccination route
(Figures 3C, S3E, and S3F).

Intrasalvational vaccination offers broad protection across serotypes

Vaccinated animals were infected on day 28 with H3N2 influenza
virus and superinfected on day 35 with serotype 2 strain D39V

<footnote>(D) HiBiT assays showed that both N and C termini are localized inside the cytoplasm. Luminescence (relative light units, RLUs) is recorded with a microplate reader.</footnote>

<footnote>(E) CRISPRi-seq in wild-type (WT) D39V and *lafB* knockout mutant (*ΔlafB*) to identify the gene interaction network.</footnote>

<footnote>(F) Fitness cost of gene depletion between WT and ΔlafB mutant.</footnote>

<footnote>(G) Growth curve of WT and ΔlafB mutant with doxycycline-inducible CRISPRi targeting *cozE* and *divIB*. Strains were grown in C + Y medium with (10 ng/ml) or without (0 ng/ml) doxycycline, and the optical density at 595 nm was measured every 10 min. Average of 3 replicates is presented.</footnote>
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>Figure 3. Intranasal vaccination with LafB protects mice against pneumococcal disease in a serotype-independent manner</figcaption></figure>

(A) C57BL/6 mice were immunized on days 0 and 14 with LafB via intranasal (flagellin-adjuvanted) or subcutaneous (alum-adjuvanted) route, with a commercial PPSV vaccine, or were untreated (mock).

(B) LafB-specific antibody response. Sera were collected (22 mock, 26 intranasal, 26 subcutaneous), and levels of LafB-specific IgG were determined by ELISA. Plots represent medians and values for individual mice. Results are pooled from 3 experiments.

(C) Spleen cells (8 mice per group) were stimulated for 72 h with LafB, and cytokine levels were determined by ELISA. Results are expressed as median and are a pool of 2 experiments. Statistical significance (*p* < 0.05, ****p < 0.0001) was assessed by one-way ANOVA Kruskal-Wallis test with Dunn's correction.

(D–G) (D) Vaccinated mice were infected with H3N2 influenza A virus on day 28 and were challenged on day 35 intranasally with S. pneumoniae. Survival with serotype 2 D39V strain (E, 5 × 10⁴ CFU; 7 mock, 12 PPSV, 12 intranasal, 12 subcutaneous) is representative of 5 experiments. Results with serotype 24F (F, 10³ CFU; 6 mock, 6 PPSV, 10 intranasal) are representative of 2 experiments, and results with serotype 15A strain (G, 5×10⁴ CFU; 6 mock, 6 PPSV, 10 intranasal) are from one experiment. (E–G) Protection was assessed by monitoring survival. Statistical significance (*p* < 0.05, ****p < 0.0001) was assessed by Mantel-Cox test.
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>**Figure 4. Th17 TRM-mediated protection in mice and LafB-specific immune responses in humans**</figcaption></figure>

(A) C57BL/6 (WT), *Rorc(t)-Gfp*<sup>TG</sup>, or *Il17a*<sup>-/-</sup> mice were immunized on days 0 and 14 with flagellin-adjuvanted LafB by intranasal route or left unvaccinated (mock) and infected with H3N2 influenza A virus on day 28. On day 35, the immune responses of virus-infected animals in (B), (D), and (E) were analyzed, or the animals were superinfected with *S. pneumoniae* strain D39V (5 x 10<sup>4</sup> CFU) to monitor survival in (C) and (F).

(B) Spleen, MdLN, and lung cells from C57BL/6 animals (n = 5 per group) were collected and stimulated for 72 h with LafB antigen. IL-17A levels in supernatant were determined by ELISA. Plots represent medians and values for individual mice. Results are representative of 2 experiments. Statistical significance (\*\*p < 0.01) was assessed by Mann-Whitney test.

(C) Vaccine protection is abolished in *Il17a*<sup>-/-</sup> mice. Results are from one experiment with 6 animals per group. Statistical significance (\*p < 0.05) was assessed by Mantel-Cox test.

(D and E) ROR γt- and IL-17A-producing lung cells in *Rorc(t)-Gfp*<sup>TG</sup> animals. Results are representative of 2 experiments with 4 mice per group. (D) Analysis of natural killer T (NKT) cells, group 3 innate lymphoid cells (ILC3), TCRγδT cells, and conventional αβ T lymphocytes. Plots represent medians and values for individual mice. Statistical significance (\*p < 0.01) was assessed by Mann-Whitney test. (E) Expression of CD69 marker on lung CD4<sup>+</sup> Th17 cells.

(F) Protection requires CD4<sup>+</sup> T cells. To this end, mice (n = 9 per group) were treated intraperitoneally on day 34 with CD4-specific depleting antibodies or control isotype, infected on day 35 with D39V, and protection was assessed. Results are representative of 2 experiments. Statistical significance (\*\*p < 0.01) was assessed by Mantel-Cox test.

(legend continued on next page)
(Figure 3D). All mice receiving mock immunization succumbed to disease after infection. Forty percent of mice vaccinated with PPSV, which includes the CPS from serotype 2, were protected against pneumococcal challenge (Figure 3E). Mice vaccinated via the i.n. route with flagellin-adjuvanted LafB outperformed both s.c.- and PPSV-vaccinated animals, with 60% mouse survival. Protection was associated with reduced weight loss during the first day following the D39V infection (Figure S4A). Moreover, surviving mice were consistently gaining weight from day 3 (Figure S4B). Mice immunized i.n. with vaccines containing LafB standalone or flagellin-adjuvanted ovalbumin were not protected (Figures S3H and S3I).

Immunoblotting showed that serum of animals vaccinated with flagellin-adjuvanted LafB recognized strains representing serotypes 1, 3, 4, 5, 9V, 11A, 15A, 19F, 23A, 23F, 24F, and 35B, corroborating the conservation of LafB (Figure S2E). Since the introduction of the CPS-based vaccines, serotypes 15A and 24F are becoming prevalent,⁸,⁵²,⁵³ which are not included in PPSV (that does contain 15B, which is cross-reactive to 15A).⁵⁴ Flagellin-adjuvanted LafB vaccination significantly protected mice against infection with the 15A and 24F strains, in contrast to mice vaccinated with PPSV, which only offered slight protection against serotype 15A (Figures 3F and 3G). Protection was supported by weight analysis (Figures S4C–S4F). LafB-vaccinated mice completely cleared pneumococcal bacteria (Figure S4G), supporting a role for LafB as a universal vaccine antigen to confer sterilizing protection.

## Protection against pneumococcal superinfection is mediated by Th17 immunity

Th17 CD4+ T lymphocytes that are functionally characterized by the expression of the retinoid orphan receptor γ t (RORγt) and the production of IL-17A, are essential for mucosal protection against pneumococcal infection.⁵⁵⁻⁵⁸ To study the mechanisms of protection, immune responses were monitored starting from day 35, a time when viral infection impairs the innate and cell-mediated immunity⁵⁹⁻⁶³ (Figure 4A). Cells isolated from spleen, MdLN, or lung from vaccinated animals secreted IL-17A after ex vivo stimulation with LafB (Figure 4B), indicating that influenza infection did not disturb the capacity of the vaccine to stimulate IL-17A. Moreover, vaccination did not alter viral replication or the virus-induced proinflammatory response, when compared with mock or s.c. immunization, as measured by the viral RNA and markers for inflammation (Figures S4H and S4I). In contrast to WT animals, Il17a-deficient mice were not protected against superinfection after i.n. vaccination (Figure 4C). Notably, the lung viral RNA quantities were unchanged in Il17a-deficient mice, and the infection remained sublethal (Figures S4J and S4K). Together, these data show that IL-17A is a major effector of immunoprotective response induced by LafB i.n. vaccination.

The main cells producing RORγt and IL-17A after i.n. vaccination and influenza virus infection were conventional CD4+ T lymphocytes

expressing TCRαβ, i.e., Th17 lymphocytes (Figures 4D and 4E). Other innate lymphocytes, such as natural killer T cells, group 3 innate lymphoid cells, or TCRγδT cells, were moderately affected. Th17 lymphocytes were associated with increased surface expression of CD69, a marker specific of tissue-resident memory (TRM) T lymphocytes in lungs.⁶⁴ Depletion of CD4+ T lymphocytes also reduced protection of the LafB vaccine against pneumococcal disease (Figure 4F). Thus, i.n. LafB vaccination induced protection dependent on lung Th17 lymphocytes with TRM features.

## Healthy human individuals develop LafB-specific immunity

To examine whether LafB might be a suitable vaccine and antigenic in humans, we screened plasma from >100 healthy donors for antigen-specific antibodies. Diphtheria toxoid was used as a positive control. The pneumococcal proteins PsaA and YchF were used as supplementary antigens. As shown in Figure 4G, individuals were all immunoreactive to diphtheria toxoid. LafB-specific antibody responses were rather low. However, 10% of individuals demonstrated a stronger LafB antibody response. The responses for PsaA and YchF were also heterogeneous and variable. Using immunoblotting, we found that immunoreactivity was associated to LafB detection (Figure 4H). Finally, peripheral blood mononuclear cells from healthy donors were stimulated with recombinant LafB or incubated with T cell stimulant phytohemagglutinin (Figure 4I). LafB significantly stimulated IFN-γ secretion, compared with controls. Together, these data indicate that LafB is antigenic in humans.

## DISCUSSION

The principal contribution of this work is the identification of a conserved intracellular membrane-associated pneumococcal antigen as a vaccine candidate effective in protection, even following influenza virus infection. The unbiased approach of antigen screening by CRISPRi in the context of superinfection defined that the protein LafB plays an essential role in pneumococcal virulence. LafB indirectly controls the levels of teichoic acids by using the lipid anchor Glc-DAG to synthesize GalGlc-DAG, similarly to TacL in the production of LTA⁴⁶,⁴⁷ (Figure S2). Indeed, LafB is important for proper cell envelope homeostasis, offering an explanation for why LafB is conserved and important for virulence. Despite its intracellular localization, LafB triggers vigorous antibody- and T cell-mediated immunity. This paradigm for antigen selection may open avenues for the discovery of virulence-associated antigens heretofore overlooked by classical approaches. In contrast to surface determinants, LafB may be exposed outside of bacteria upon the production of extracellular vesicles,⁶⁵ lysis, or autolysis. Indeed, during colonization, pneumococci establish biofilms that consist of a matrix of lysed bacteria.⁶⁶ A subset of healthy individuals have LafB-specific

<footnote>(G) Plasma samples from healthy donors (n = 127) were analyzed by ELISA for reactivity specific to the following antigens: diphtheria toxoid (DT) and the pneumococcal proteins LafB, PsaA, and YchF. Plots represent medians and values for individual donors.</footnote>

<footnote>(H) Plasma samples (n = 4 per group) with low and high absorbance at 450 nm in ELISA were analyzed by immunoblotting. LafB was separated by SDS-PAGE and transferred to a membrane before probing with plasma.</footnote>

<footnote>(I) Peripheral blood mononuclear cells from healthy donors (n = 6) were stimulated for 5 days with LafB or phytohemagglutinin (PHA) or were untreated. Secretion of IFN-γ was determined by ELISA. Statistical significance (*p* < 0.05, **p* < 0.001) was assessed by one-way ANOVA Kruskal-Wallis test with Dunn's correction.</footnote>
IgG in their serum, indicating that LafB is also antigenic in humans. It should be noted that LafB is highly conserved in pneumococci (Figure S1), and to a lesser extent in members of the mitis groups, meaning that LafB-specific antibodies do not strictly indicate previous pneumococcal carriage or infection. Unraveling the immune circuits underlying the respiratory protective response is an important question for future research. In addition, it would be interesting to test the efficacy of i.n. vaccination against primary pneumonia caused by pneumococcus.

Multiple lines of evidence show that Th17 lymphocytes are instrumental for protecting the respiratory mucosa against pneumococcal infection.⁵⁵⁻⁵⁸,⁶⁷,⁶⁸ Moreover, preceding influenza virus infection may blunt IL-17 production by γδT cells in response to S. pneumoniae.⁶⁹ Cross-protection against pneumococcal diseases after recovery from a primary infection is mediated by memory Th17 cells, but the antigenic determinants remained to be defined.⁵⁸ Interestingly, memory Th17 responses elicited by pneumococcal infection can overcome viral-driven Th17 inhibition and provide cross-protection against different serotypes during co-infection with influenza virus. Thus, a vaccine that drives Th17 responses could mitigate disease caused by co-infection.⁷⁰ LafB may constitute such a prototypic cross-protective antigen. Recent studies highlighted how lung Th17 cells differentiate into TRM that persist in tissue, promote long-term protection against pathogens,⁵⁷,⁶⁴ and are less prone to collapse in the context of immunosuppression.⁵⁷ Stimulation of lung Th17 lymphocytes and TRM may explain the inferior protective capacity of systemic route of immunization. Similar observations were made for COVID-19 vaccination in which antibody levels do not correlate with better disease outcome, particularly in older individuals.⁷¹ A next generation of vaccines could incorporate CPS conjugated to LafB for intramuscular priming, followed by an i.n. boost with adjuvanted LafB. This vaccination regimen is poised to induce (1) high-affinity opsonizing circulating antibodies against CPS and (2) lung-resident memory Th17 cells. These two arms of adaptive immunity might operate synergistically in systemic and mucosal compartments, contributing to reduced carriage and immunoprotection. The use of mucosal adjuvants to potentiate lung protective immunity is an expanding field of research that will lay the foundation of a new generation of vaccines against respiratory pathogens.⁵⁷,⁷²,⁷³

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

* Materials availability

* Data and code availability

* EXPERIMENTAL MODELS AND SUBJECT DETAILS

* Bacterial strains and culture conditions

* Mouse models

* Murine superinfection assays

* Human samples

## METHODS DETAILS

* Phase contrast and fluorescence microscopy

* Split-luciferase HiBiT-tag detection system assay

* Bacterial genomic DNA extraction and CRISPRi-seq

* LafB synthetic lethal screening by CRISPRi-seq

* Mice vaccination

* Antigen-specific immune responses

* Flow cytometry analysis

* Analysis of plasma and PBMC from healthy individuals

* Rabbit LafB antiserum

* Western blot and immunoblot

* Doxycycline stock

* Purification of LafB and YchF protein from E. coli

* Biochemical characterization of the LafB glycosylation activity

* Bacterial colonization

* Viral RNA quantification

* Proinflammatory gene expression

* Strain construction

* Antisera production in rabbit used for western blotting

* QUANTIFICATION AND STATISTICAL ANALYSIS

* ADDITIONAL RESOURCES

* DATA AND MATERIALS AVAILABILITY

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j. chom.2024.02.002.

## ACKNOWLEDGMENTS

We thank Mara Baldry and Charlotte Costa for assistance with animal experiments, Frédéric Wallet for clinical isolates, Loic Coute for *II17a*⁻¹ mice, Delphine Cayet for ELISA, Guillaume Lefebvre and Julie Démaret for discussions, Cyrille Grandjean for PsaA, and Jingwei Xu for RoseTTAFold predictions. We thank UNIL's Metabolomics and Genomics Technologies Facility, UAR2014-US41-PLBS, Bioimaging Center Lille, Flow Cytometry Core Facility, and the PLETHA animal facility. This work was supported by an ERC consolidator grant (771534) and Swiss National Science Foundation grants (310030_192517, 310030_200792, 51NF40_180541, and IZSEZ0_213879) to J.-W.V.; Inserm, Institut Pasteur de Lille, Université de Lille, and the Structure Fédérative de Recherche grant PneumoVac to L.V.M.; the project FAIR, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 847786 to J.-C.S.; a National Nature Science Foundation of China grant (82270012), the Science and Technology Project of Shenzhen (JCYJ20220818095602006), and the Shenzhen University 2035 Program for Excellent Research (86901-00000216) to X.L.

## AUTHOR CONTRIBUTIONS

Experimentation, X.L., L.V.M., D.S., L.M., V.A.D.d.S., J.D., F.P.B., M.T., and J.O.; study design and analysis, X.L., L.V.M., L.M., V.d.B., S.G., and J.-W.V.; writing – original draft, X.L., L.V.M., J.-C.S., and J.-W.V.; writing – review & editing, X.L., V.N., J.-C.S., and J.-W.V.

## DECLARATION OF INTERESTS

X.L., L.V.M., F.P.B., J.-C.S., and J.-W.V. have filed patent application WO 2023/006825 on aspects of this work. J.-C.S. is the inventor of patent WO2009156405, describing recombinant flagellin as an adjuvant.

Received: January 24, 2023

Revised: December 6, 2023

Accepted: February 5, 2024

Published: February 27, 2024
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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>InvivoMab rat anti-mouse CD4 (GK1.5 monoclonal antibody, Bio X cell)</td><td>Euromedex</td><td>BX-BE003-1-25MG</td></tr><tr><td>InvivoMab rat IgG2b isotype control (LTF-2 monoclonal antibody, Bio X cell)</td><td>Euromedex</td><td>BX-BE0090-25MG</td></tr><tr><td>HRP-conjugated goat anti-mouse IgM (Southern Biotech)</td><td>Clinisciences</td><td>1020-05; RRID: AB_2794201</td></tr><tr><td>HRP-conjugated goat anti-mouse IgG (Southern Biotech)</td><td>Clinisciences</td><td>1030-05; RRID: AB_2619742</td></tr><tr><td>HRP-conjugated goat anti-mouse IgA (Southern Biotech)</td><td>Clinisciences</td><td>1040-05; RRID: AB_2714213</td></tr><tr><td>Rabbit antiserum against LafB</td><td>Eurogentec</td><td>N/A</td></tr><tr><td>HRP conjugate goat-anti-mouse IgG</td><td>Promega</td><td>Cat#W4021; RRID: AB_430834</td></tr><tr><td>HRP conjugate goat-anti-rabbit IgG</td><td>Abcam</td><td>Cat#AB205718; RRID: AB_2819160</td></tr><tr><td>HRP conjugate goat-anti-human IgG</td><td>Sigma-Aldrich</td><td>Cat#A0170; RRID: AB_257868</td></tr><tr><td>TCRd-PerCP-eFluor710</td><td>Thermo Fisher Scientific</td><td>46-5711-82; RRID: AB_2016707</td></tr><tr><td>CD45-AF700</td><td>Biolegend</td><td>103128; RRID: AB_493715</td></tr><tr><td>CD19-APC-Cy7</td><td>Biolegend</td><td>115530; RRID: AB_830707</td></tr><tr><td>Gr1-APC-Cy7</td><td>Biolegend</td><td>108424; RRID: AB_2137485</td></tr><tr><td>TCRb-BV421</td><td>BD biosciences</td><td>562839; RRID: AB_2737830</td></tr><tr><td>CD90.2-BV510</td><td>Biolegend</td><td>105335; RRID: AB_2566587</td></tr><tr><td>NKp46-biotine</td><td>Biolegend</td><td>137616; RRID: AB_11219387</td></tr><tr><td>CD11b-biotine</td><td>Biolegend</td><td>101204; RRID: AB_312786</td></tr><tr><td>CD11c-biotine</td><td>Biolegend</td><td>117304; RRID: AB_313773</td></tr><tr><td>CD127-PE-Cy7</td><td>Thermo Fisher Scientific</td><td>25-1271-82; RRID: AB_469649</td></tr><tr><td>CD69-PE</td><td>Thermo Fisher Scientific</td><td>12-0691-81; RRID: AB_465731</td></tr><tr><td>CD103-BV711</td><td>Biolegend</td><td>121435; RRID: AB_2686970</td></tr><tr><td>IL-17A-APC</td><td>Miltenyi Biotec</td><td>130-112-010; RRID: AB_2652362</td></tr><tr><td colspan="3"><strong>Bacterial and Virus Strains</strong></td></tr><tr><td>Streptococcus pneumoniae</td><td>Laboratory stock, UK Health Security Agency</td><td>NCTC 14078</td></tr><tr><td>(Other bacterial strains are listed in Supplementary Table S2)</td><td>This paper</td><td>N/A</td></tr><tr><td>Murine-adapted H3N2 influenza A virus</td><td>Dr Mustapha Si-Tahar (University of Tours)</td><td>strain Scotland/20/74</td></tr><tr><td colspan="3"><strong>Human samples</strong></td></tr><tr><td>Blood from anonymous healthy donors from France</td><td>French Public Health Code</td><td>EFS contract no. NT/18/2016/200</td></tr><tr><td colspan="3"><strong>Chemicals, Peptides, and Recombinant Proteins</strong></td></tr><tr><td>D-luciferine</td><td>Synchem</td><td>CAS:115144-35-9</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>Cat#A1120</td></tr><tr><td>NucleoSpin Microbial DNA</td><td>Macherey-Nagel</td><td>Cat#740235.50</td></tr><tr><td>Nano-Glo® HiBiT Extracellular Detection System</td><td>Promega</td><td>Cat#N2420</td></tr><tr><td>Flagellin FliC<sub>Δ174-400</sub></td><td>Laboratory production</td><td>N/A</td></tr><tr><td>Imject™ Alum Adjuvant</td><td>ThermoFisher</td><td>Cat#77161</td></tr><tr><td>PPSV vaccine Pneumovax®</td><td>MSD</td><td>Pneumovax®</td></tr><tr><td>LafB protein</td><td>This paper</td><td>N/A</td></tr><tr><td>YchF protein</td><td>This paper</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>PsaA protein</td><td>Dr. Cyrille Grandjean from UMR CNRS 6286 in Nantes, France</td><td>N/A</td></tr><tr><td>Diphteria toxoid</td><td>Instituto biologico argentino S.A.I.C</td><td>DAM 049</td></tr><tr><td>Brefeldin A</td><td>Merck</td><td>B5936-200UL</td></tr><tr><td>Collagenase VIII</td><td>Merck</td><td>C2139-5G</td></tr><tr><td>PMA</td><td>Merck</td><td>P8139-1MG</td></tr><tr><td>Ionomycine calcium salt 1mM</td><td>Merck</td><td>I3909-1ML</td></tr><tr><td>PhytoHemagglutinin-L</td><td>Fisher Scientific</td><td>15556286</td></tr><tr><td>SepMate™</td><td>StemCell</td><td>85450</td></tr><tr><td>1,2-Diacyl-3-alpha-D-glucosyl-sn-glycerol</td><td>Avanti</td><td>CAS: 2021179-21-3</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt</td><td>Sigma</td><td>CAS: 67254-28-8</td></tr><tr><td>UDP-α-D-Galactose</td><td>Merck Millipore</td><td>CAS: 137868-52-1</td></tr><tr><td>NucleoSpin RNA II kit</td><td>Macherey-Nagel</td><td>740955.250</td></tr><tr><td>Superscript II Reverse Transcriptase</td><td>Invitrogen</td><td>18064014</td></tr><tr><td>High-Capacity cDNA Archive Kit</td><td>Applied Biosystems</td><td>4368813</td></tr><tr><td>Takyon™ Low ROX SYBR</td><td>Eurogentec</td><td>UF-LSMT-B0710</td></tr><tr><td>Desoxyribonuclease I</td><td>Sigma-Aldrich</td><td>10104159001</td></tr><tr><td>Intracellular Fixation & Permeabilization kit</td><td>eBiosciences</td><td>88-8824-00</td></tr><tr><td>TMB</td><td>BD Bioscience</td><td>555214</td></tr><tr><td>Rompun 2% (xylazine)</td><td>Centravet</td><td>ATC: QN05CM92</td></tr><tr><td>Imalgen 1000 (ketamine)</td><td>Centravet</td><td>ATC: QN01AX03</td></tr><tr><td>Isoflurin</td><td>Centravet</td><td>ATC: QN01AB06</td></tr><tr><td>Doxycycline hyclate</td><td>TCI</td><td>D4116-5G</td></tr><tr><td>Streptavidin-BV605</td><td>biolegend</td><td>405229</td></tr><tr><td>Biorad protein quantification kit</td><td>Biorad</td><td>Cat#5000002</td></tr></tbody></table>

Deposited Data

<table><tr><td>Sequencing output (Fastq files)</td><td>This paper</td><td>PRJNA895037</td></tr><tr><td>Experimental Models: Organisms/Strains</td><td></td><td></td></tr><tr><td>Mouse: C57BL/6J</td><td>Janvier Labs</td><td>C57BL/6JRj</td></tr><tr><td>Mouse: <i>Rorc(γt)-Gfp</i><sup>TG</sup></td><td>housing</td><td><i>Rorc(γt)-Gfp</i><sup>TG</sup></td></tr><tr><td>Mouse: <i>I17a</i><sup>-/-</sup></td><td>housing</td><td><i>I17a</i><sup>-/-</sup></td></tr><tr><td>Mouse: BALB/c</td><td>Janvier Labs</td><td>BALB/cJRj</td></tr></table>

Oligonucleotides

<table><tr><td>Oligos for this study (See Table S3)</td><td>Sigma or Eurogentec</td><td>N/A</td></tr></table>

Recombinant DNA

<table><tr><td>pLIBT7_A_lafB-CPDHisOld</td><td>This paper</td><td>N/A</td></tr><tr><td>sgRNA library</td><td>Addgene</td><td>#170432</td></tr><tr><td>pLIBT7_A_ychF-CPDHisOld</td><td>This paper</td><td>N/A</td></tr><tr><td>pPEPZ-sgRNAclone</td><td>Addgene</td><td>#141090</td></tr></table>

Software and Algorithms

<table><tr><td>Prism v8.0</td><td>GraphPad Software</td><td>https://www.graphpad.com/</td></tr><tr><td>ImageJ v2.0</td><td>National Institutes of Health</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>R v4.1.1</td><td>The R Foundation for Statistical Computing</td><td>https://www.r-project.org/</td></tr><tr><td>Illustrator CC</td><td>Adobe</td><td>https://www.adobe.com</td></tr><tr><td>2FAST2Q (v.2.4.1)</td><td>NA</td><td>https://veeninglab.com/2fast2q</td></tr><tr><td>FlowJo</td><td>FlowJo</td><td>https://www.flowjo.com</td></tr></table>
RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jan-Willem Veening (jan-willem.veening@unil.ch).

Materials availability

The sgRNA library is available through Addgene (#170432). The generated rabbit antiserum against LafB may be available from the lead contact under a material transfer agreement.

Data and code availability

* The fastq files generated from sequencing are uploaded to the Sequence Read Archive on NCBI with accession number PRJNA895037.

* This work did not use or generate new code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Bacterial strains and culture conditions

S. pneumoniae strains were grown in liquid semi-defined C+Y medium, pH = 6.8⁷⁴ at 37C from a starting optical density (OD₆₀₀nm) of 0.01 until the appropriate OD. Transformation of S. pneumoniae was performed as described previously.³³ Transformants were selected on Columbia agar with 5% sheep blood with antibiotics (100 µg/ml spectinomycin, 250 µg/ml kanamycin, 1 µg/ml tetracycline, 40 µg/ml gentamycin, 0.05 µg/ml erythromycin). E. coli strains were grown with LB medium or LB agar with appropriate concentrations of antibiotics (100 µg/ml ampicillin, 100 µg/ml spectinomycin, or 50 µg/ml kanamycin). All strains, plasmids and primers used are listed in Tables S2 and S3 within the supplementary information.

Mouse models

Mice experiments complied with national, institutional and European regulations and ethical guidelines, and were approved by the Institutional Animal Care and Use Committee (animal facility agreement D59-350009, Institut Pasteur de Lille, protocol reference: APAFIS#16966, 201805311410769_v3). Six to eight weeks old male C57BL/6JRj, *Rorc(γt)-Gfp<sup>TG</sup>, Il17a<sup>-/-</sup> mice were purchased from Janvier Laboratories (Saint Berthevin, France) or bred and maintained in individually ventilated cages (Innorack® IVC Mouse 3.5) and handled in a vertical laminar flow biosafety cabinet (Class II Biohazard, Tecniplast). Male mice were used since they are larger and can better accommodate the volume of instillation of viral and bacterial inoculum than females. All infections were performed in an animal biosafety level 2 facility. For depletion of CD4⁺ cells, mice received an intraperitoneal injection of 200 µg of GK1.5 monoclonal antibody (rat anti-mouse CD4, Bio X cell) or control isotype on day 34.

Murine superinfection assays

Infections were performed by intranasal (i.n.) route in mice that were previously anesthetized by intraperitoneal (i.p.) injection of 1.25 mg of ketamine and 0.25 mg of xylazine in 200 µL of PBS. Male C57BL/6JRj mice were infected i.n. on day 0 with 50 plaque-forming units (PFU) of the pathogenic murine-adapted H3N2 influenza A virus strain Scotland/20/74 in 30 µL of PBS as previously described.³⁴ On day 7, mice were infected i.n. with S. pneumoniae strains using 5 × 10⁴ CFU of the frozen working stocks diluted in 30 µL of PBS or CRISPRi library using 1.5 × 10⁵ CFU. For CRISPRi experiments, doxycycline (5mg/kg in 200 µL PBS) or PBS (200 µl) were injected intraperitoneally (i.p.) at 1h before and 9 h after S. pneumoniae infection. At 24 h post-pneumococcal infection, mice were euthanized by i.p. injection of 5.47 mg of sodium pentobarbital. Lungs and spleen were collected in 1 ml of PBS, homogenized with a T-25 digital Ultratrurrax® (IKA) to evaluate CFU count or extract genomic DNA. In some experiments, mouse survival and weight were recorded every day for 10 days after S. pneumoniae infection.

Human samples

Blood samples obtained from healthy donors from France to prepare plasma. Blood donation in France is an act of citizenship, sol-darity and freedom, and always conducted under strict anonymity by the "Etablissement Français du Sang (EFS "Nord de France", Lille). EFS is the French blood establishment, the sole civil operator of blood transfusion in France. Data on age, sex, ancestry, race, ethnicity, and socioeconomic status are thus not collected. Written informed consents for the use of blood for research objectives were obtained from the donors under EFS contract no. NT/18/2016/200 with respect to Decree no. 2007-1220 (articles L1243-4, R1243-61 and following) dated August 10, 2007, of the French Public Health Code. The use of human samples was approved by the French Ministry of Education and Research under the agreement DC 20152575.
## METHODS DETAILS

### Phase contrast and fluorescence microscopy

S. pneumoniae cells were grown in C+Y medium pH = 6.8 at 37°C to an OD$_{595nm}$ = 0.1 without any inducer and diluted 100-fold in fresh C+Y medium supplemented with 100 µM IPTG. After 1 h of incubation at 37°C in a 5% CO$_{2}$ incubator, bacteria were harvested from 1 ml culture by centrifugation at 8000 g, 2 min. The pellet was resuspended in 50 µl PBS, and 0.5 µl of the suspension was spotted onto a PBS agarose pad on microscope slides. Visualization of GFP was performed as described previously.⁷⁵

### Split-luciferase HiBit-tag detection system assay

The assay was performed with the Nano-Glo® HiBit Extracellular Detection System (Promega). The gene sequence encoding the HiBit tag was inserted to the 5' or 3' end of *lafB* coding sequence by cloning, and the HiBit-tagged *lafB* gene was driven by an IPTG inducible promoter and introduced into the chromosome of *S. pneumoniae* Δ*lafB* mutant. Construction of the mutants is described in the supplementary methods. Note that *lafB* is also known as *cpoA*.³⁷ The HiBit tag is an 11 amino acid peptide, which can bind to a larger subunit and form a complex with luciferase activity.⁷⁶ In this assay, the LgBit subunit can be added into the reaction mixture, but it cannot go through the membrane. So, only when the HiBit tag is exposed to the outside of the cell membrane, luminescence signal can be detected. The S. pneumoniae mutants carrying N- or C-terminal Hibit tagged LafB were grown in C+Y acid medium, pH = 6.8, at 37°C until OD600 = 0.3. The culture was then diluted 1:10 into fresh C+Y acid medium with or without 1 mM IPTG for the induction of protein expression. When the OD600 of bacterial culture reached 0.3, the culture was split into two groups: one group was the lysed cell group, in which the bacteria were lysed by addition of 16 µg/ml phage lysin cpl-1,⁷⁷ and the other group was the intact cell group, without any lysis step performed. The luciferase assay was performed according to the manufacturer's instructions with the lysed and intact cells. 50 µl of bacterial culture was mixed with 50 µl of reaction reagent of the kit in a black 96-wells plate. Bioluminescence was quantified on a Tecan Infinite 200 PRO luminometer at 37°C. Bioluminescence was measured right after the reagent addition. Four replicates for each condition were performed.

### Bacterial genomic DNA extraction and CRISPRi-seq

Lung homogenates were mixed with deoxyribonuclease I (10 µg/mL, Sigma-Aldrich) for 10 min at room temperature and filtered through 100 µm meshes. The filtrate underwent centrifugation at 16,000 g and the pellet was used for bacterial gDNA extraction using the NucleoSpin Microbial DNA kit (Macherey-Nagel). For bacterial lysis, 6 cycles of 45 s agitation at a speed of 6 m/s in a FastPrep-24™ homogenizer (MP Biomedicals) were used. DNA was quantified in a NanoDrop spectrophotometer (ThermoFisher) The gDNA was used as template in the one-step PCR to prepare the amplicon libraries for Illumina sequencing as described.³³ Sequencing was performed on an Illumina MiniSeq platform using a 54 dark cycle custom recipe.³³ Raw sgRNA counts were obtained from the fastq files using 2FAST2Q (v.2.4.1) with default settings (PHRED score of minimally 30 and 1 mismatch allowed).⁷⁸ Mouse samples PBS 1 and 6, and DOX 1, 5 and 6 were excluded from downstream analyses, on grounds of low pool diversity even without CRISPRi induction (PBS 6), and bottleneck effects (PBS 1, DOX 1, 5, 6). Samples were excluded if >5% of the strains dropped out of the pool without CRISPRi induction, or if the estimated bottleneck size was smaller than three times the pool diversity. Bottleneck sizes were estimated as before,³⁴ with pneumococcal generation time assumed to be 108 min, time of growth 24h, and the total population size at the start $1.5×10^6$ CFU. Differential fitness analyses were performed using DESeq2 (v.1.34.0) in R (v.4.1.1), with hypothesis testing threshold values of an absolute $log_2FC>1$ and $alpha<0.05$.

### LafB synthetic lethal screening by CRISPRi-seq

A *lafB* knockout strain (VL4017) was constructed in the background of Plac-dcas9 containing strain DCI23⁷⁹ as described in the supplementary methods. The pneumococcal sgRNA library (Addgene #170432) was transformed into the resulting strain. Genome-wide fitness quantification via CRISPRi-seq was then performed in both the wild-type and the *lafB* deletion mutant in parallel. Treatment of the libraries was performed as described before.³⁴ Specifically, the induction treatment with 1 mM IPTG lasted for 21 generations in C+Y medium, and the control group without induction was set up. The bacteria were collected after treatment followed by genomic DNA isolation. CRISPRi-seq was performed as described previously.³³ Raw sgRNA counts were obtained from the fastq files using 2FAST2Q (v.2.4.1) with default settings.⁷⁸ Differential fitness analyses were performed using DESeq2 (v.1.34.0) in R (v.4.1.1), with hypothesis testing threshold values of an absolute $log_2FC>1$ and $alpha<0.05$.

### Mice vaccination

Mice were vaccinated by the intranasal (i.n.) or subcutaneous (s.c.) route at days 0 and 14. A single vaccine dose per animal contained 20 µg LafB in combination with flagellin FliC$_{Δ174-400}$ (2.5 µg) for intranasal vaccination (30µl) or Imject™ Alum (ThermoFisher) for subcutaneous vaccination (100µl). The PPSV vaccine, i.e. Pneumovax® from MSD was used ($1/_{6}$ human dose/mice). This represents a dose of 4.17 µg of each capsular polysaccharide per animal (including the serotype 2 capsule produced by the strain D39V). This high dose was chosen to promote optimal anti-capsule antibody response. Other studies have used 1/5th to 1/200th of the human dose.¹⁶,⁸⁰ For LafB, the dosage was based on similar doses than for other antigens used in vaccination studies. Usually, 10 to 50 µg of antigens (molecular mass between 30-60 KDa) are used in each dose of vaccination experiments in mice. In our study, LafB that is a 40KDa antigen was used at dose of 20 µg per vaccination. Intranasal vaccination was performed under slight anesthesia by gaseous isoflurane (Axience). Antibody and T cell responses were analyzed on blood, spleen, lung and MdLN at day 28 or at day 35, i.e., 7 days after the influenza virus infection.
Antigen-specific immune responses

LafB-, YchF-, PsaA- or Diphteria toxoid (DT)-specific antibodies in serum were assessed by ELISA. Plates were prepared by absorp-
tion of LafB, YchF, PsaA or DT (1 µg/ml in carbonate buffer), overnight at 4°C on Maxisorp microplates (Nunc), and 1 h blocking at room-
temperature with 1% dried milk in PBS. ELISA plates were incubated for 1 h at room temperature with serial dilutions of the serum sam-
ples. Primary antibody binding was revealed with subsequent incubations with HRP-conjugated goat anti-mouse IgG, IgM or IgA
(Southern Biotech) and TMB (BD Bioscience); and measured using a microplate reader at 450/570 nm wavelength. LafB-specific
T cell responses were analyzed in spleen, MdLN or lungs. Cells (1×10⁶) were incubated for 72h with RPMI 1640 with 10% fetal calf
serum, 2mM glutamine, 1mM sodium pyruvate, 10mM HEPES, non-essential amino acids, 100U/100µg Penicillin-Streptomycin
and stimulated or not with LafB antigen (50 µg/mL) to measure secretion of IL-13, IL-17A, IL-22, or IFN-γ by ELISA.

Flow cytometry analysis

Lungs were digested with collagenase IA (Sigma, 1 mg/ml) and DNase I (Sigma, 40µg/ml) during 15 min at 37°C. Cells were separated on Percoll 20% and stained for TCRd-PerCP-eFluor710, CD45-AF700, CD19-, Gr1-APC-Cy7, TCRb-BV421, CD90.2-BV510, NKp46-, CD11b-, CD11c-BV605, CD103-BV711, CD69-PE or CD127-PE-Cy7 (Becton Dickinson or Biolegend). Cells were incubated 4h with Brefeldin A (10µg/ml), PMA (25ng/ml) and ionomycin (500ng/ml) and processed for intracellular staining using the kit Intracellular Fixation & Permeabilization (eBiosciences) and IL-17A-APC or control isotype (REAffinity, Miltenyi Biotec). Data were collected on a BD LSR Fortessa and analyzed with FlowJo software.

Analysis of plasma and PBMC from healthy individuals

Plasma (n = 127) and whole blood cells (n = 6) were collected from healthy donors. LafB-, PsaA-, and YchF-specific antibodies were analyzed by ELISA as described above using peroxydase-conjugated goat anti-human IgG antibodies (Sigma-Aldrich). PBMC were purified from blood samples using the SepMate™ (StemCell) as described by the manufacturer. PBMC were cultured (1x10⁶) for 5 days with RPMI 1640 with 10% fetal calf serum (FCS), 2mM glutamine, 1mM sodium pyruvate, 10mM HEPES, non-essential amino acids, 100U/100µg Penicillin-Streptomycin and stimulated or not with LafB (1µg/ml or 10µg/ml) or Phytohemagglutinin (PHA, 1µg/ml) to measure secretion of IFN-γ by ELISA.

Rabbit LafB antiserum

The rabbit antiserum against LafB was produced by Eurogentec with the speedy 28-day program. The protocol uses a non-Freund adjuvant, and the immunization schedule includes 4 injections on days 0, 7, 10 and 19. 100 µg/injection of tag-free LafB protein was used for the immunization. Before the first injection, 1 pre-immune bleed and ELISA were performed to make sure the absence of antibodies against LafB in the naive rabbit. Then on day 21, 1 medium bleed was performed to test the production of IgG with ELISA, and the final bleed was performed on day 28.

Western blot and immunoblot

To test the antisera of immunized rabbit or mouse. The S. pneumoniae strains were grown in 5 ml of acid C+Y medium to OD600 0.3. The cells were harvested by centrifugation at 8000 g, 5 min. The supernatant was removed, and the pellets were resuspended with cold TE buffer (10 ml of 1 M Tris-HCl pH7.5, 2 ml of 0.5 M EDTA pH8, 88 ml of MQ water, to final 100 ml) for 2 times. Resuspend the pellet in 200 µl of TE buffer, and then break down the bacteria by sonication (1s pulse on, 1s pulse off, Amp 40%) until the solution becomes clear. The total protein concentration was quantified with Biorad protein quantification kit (Biorad, Cat. 5000002), and then all the samples were normal-ized to the same protein concentration by dilution with TE buffer, which was around 1 mg/ml. 100 µl of cell lysate was mixed with 100 µl of 2×SDS loading buffer, and then boiled at 95°C for 10 min. The samples underwent centrifugation at 15,000 g for 5 min, and then 10 µl of each sample was loaded into one well of a 12% SDS-PAGE (Bio-Rad, Cat. 4561046). 5 µl of PageRuler Plus Prestained Protein Ladder (Fisher, Cat.e 26619) was loaded as marker. The protein samples were transferred onto a PVDF membrane. The PVDF membrane with protein samples was blocked with 5% skim milk (PanReac Applichem, A0830) in PBST (PBS pH7.4 with 0.1% Tween-20) under room temperature for 2 h. The antisera of rabbit or mouse was diluted 1:500 in PBST and then added onto the membrane for 1 h incubation at room temperature. The membrane was then washed 3 times, 5 min each time. The secondary antibodies HRP conjugate goat-anti-mouse IgG (Promega, Cat. W4021; 1:2500 dilution in PBST) or HRP conjugate goat-anti-rabbit IgG (Abcam, Cat. AB205718; 1:5000 dilution in PBST) was added onto the membrane incubated with mouse or rabbit antiserum as the first antibody, respectively. The secondary antibody was incubated with the membrane at room temperature for 1 h, followed by 3 times of washing, 10 min each time. Detection is performed using the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo scientific, Cat. 34579), and the visualization is performed with the FusionCapt Advance FX7 (Witec AG). To test human plasma, purified LafB antigen (500 ng) was loaded on a 4 to 20% SDS-PAGE, transferred to a nylon membrane, and probed with patient plasma (1:100 dilution) overnight at 4°C. The blot was revealed with a horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (1:10,000 dilution, Sigma-Aldrich) 1h at room temperature and visualized with an enhanced chemiluminescence-based detection kit (West Pico PLUS, Thermo Scientific).
## Doxycycline stock

Doxycycline hyclate (TCI Europe) stock solutions were prepared in PBS at a concentration of 10 mg/mL, filtered through 0.22 µm sterile membranes, aliquoted and stored at -80°C. Fresh dilutions were prepared from frozen stocks and the doxycycline free base concentration was corrected using the conversion factor 0.8.

## Purification of LafB and YchF protein from E. coli

The *lafB* and *ychF* genes were cloned with a CPD tag into vector pLIBT7-A and maintained by *E. coli* DH5α. The recombinant vector was transformed into *E. coli* strain BL21 freshly for protein expression. To induce the expression of LafB or YchF protein in *E. coli* BL21, the strain with the recombinant vector was cultured in 500 ml of buffered TB medium to OD600nm ~0.6 at 37°C, 200 rpm. To prepare the buffered TB medium, first make the 10× Phosphate-buffered saline (2.4 g of KH₂PO₄ and 12.5 g of K₂HPO₄ in 1 L MQ water, autoclave at 121°C, 15 min), and then make the TB medium (24 g of tryptone, 48 g of yeast extract, and 10 ml of glycerol in 900 ml of MQ water, autoclave at 121°C, 15 min), and finally 900 ml of TB medium was mixed with 100 ml of the 10× Phosphate-buffered saline to make 1 L of buffered TB.

The culture was chilled to 16°C when reached OD600nm ~0.6, followed by addition of 0.5 mM IPTG (Isopropyl β-d-1-thiogalactopyranoside) to induce the expression of the recombinant protein with CPD tag overnight (for ~14 h). The bacteria were collected by centrifugation at 4°C, 5000 g. The pellets were resuspended with 75 ml of buffer (50 mM Tris-HCl, pH = 7.5, 300 mM NaCl, 5% Glycerol, 25 mM Imidazole, 5 mM 2-mercaptoethanol, 1 mM PMSF, 750 Units of nuclease). *E. coli* cells were lysed by sonication. Cell lysates underwent centrifugation at 18,000 g, at 4°C for 30 min. The supernatant was then collected for protein purification with cobalt beads. The protocol for purification of the CPD tagged protein is similar to the protocol published previously.⁸¹ Specifically, the supernatant was directly loaded onto cobalt beads, followed by washing with buffer (20 mM Tris, 100 mM NaCl) to remove the nonspecific bindings. We then used 25 ml of elution buffer (20 mM Tris, 100 mM NaCl with 2 mM inositol hexakisphosphate (InsP6)) to elute the protein. Addition of InsP6 activates the protease activity of CPD and the tag is cleaved off, so the final purified protein is tag free. The elution of LafB protein was further purified with Heparin column, and a gradient washing was made by mixing with buffer A1 (20 mM Tris, 100 mM NaCl) and buffer B1 (20 mM Tris, 1 M NaCl). The purified LafB was checked by SDS-PAGE (Figure S2A). Protein YchF was purified in similar way, except that the Hi Trap Q HP anion exchange chromatography column was used for further purification.

## Biochemical characterization of the LafB glycosylation activity

Reactions were done with PBS as the principal buffer solution. 30 uL total reaction volumes were set up to contain 10 mM lipid mixture consisting of 1 mM MGlcDAG (1,2-Diacyl-3-alpha-D-glucosyl-sn-glycerol, Avanti) and 9 mM DOPG (1,2-Dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, Sigma) as well as 1 mM UDP-α-D-Galactose (Merck Millipore). Volumes of purified LafB to desired concentration were added to start reactions. Samples were incubated at 28°C for 30 min before being quenched by the addition of Methanol to a final concentration of 80%. Samples were flash frozen and kept at -80°C until analysis by mass spectrometry.

## Bacterial colonization

For quantification of bacteria, lungs and spleen were collected 13 days after infection and homogenized in PBS. Viable counts (colony forming unit [CFU]) were determined by plating serial dilutions onto 5% blood-agar plates.

## Viral RNA quantification

Total RNA was extracted with the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). For H3N2 RNA detection, 500 ng of total lung RNA were reverse transcribed with Superscript II Reverse Transcriptase (Invitrogen) in the presence of IAV specific primers targeting the segment 7 which encodes the matrix protein 1 (M1) (5' TCTAACCGAGGTCGAAACGTA 3'). The cDNA was amplified by Taqman real-time PCR using Taqman probe FAM-TTTGTGTTCACGCTCACCGTGCC-TAMRA with forward primer: AAGACCAATC CTGTCACCTCTGA and reverse primer: CAAAGCGTCTACGCTGCAGTCC. A plasmid coding the M1 gene was serially diluted to establish a standard curve (Ct values / plasmid copies). Equivalent of 12.5 ng of total lung RNA was thoroughly used to determine the level of M1 RNA in the lung of infected animals by absolute quantification.⁸²

## Proinflammatory gene expression

Total RNA was extracted with the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). Total RNA was reverse-transcribed with the High-Capacity cDNA Archive Kit (Applied Biosystems). cDNA was amplified using Takyon™ Low ROX SYBR (Eurogentec). Relative mRNA levels were determined by comparing (a) the cycle thresholds (Ct) for the gene of interest and 2 calibrator genes (ΔCt), *Actb* and *B2m*, and (b) 2-ΔCt values for vaccinated group compared with mock group. The specific primers are CGTCATCCATGGC GAACTG/GCTTCTTTGCAGCTCCTTCGT (*Actb*), TGGTCTTTCTGGTGCTTGTC/GGGTGGCGTGAGTATACTTGAA (*B2m*), CCCTCAA CGGAAGAACCAAA/CACATCAGGTACGATCCAGGC (*Cxcl2*), CTCCAGAAGGCCCTCAGACTAC/GGGTCTTCATTGCGGTGG (*Il17a*), GTTCTCTGGGAAATCGTGGAAA / GTTCTCTGGGAAATCGTGGAAA (*Il6*), and TCAGCAACAGCAAGGCGAAA / CCGCTTCCTG AGGCTGGAT (*Ifng*).
<figure><img src="image_45.png" /></figure>

Strain construction

Parent strains for IPTG- or tet-inducible systems

The prsA-Iacl-GmR or prsA-Iacl-tetR-GmR fragments were amplified from S. pneumoniae strain D-LT-PEP9Ptela⁸³ with primers OVL1694 and OVL1695. The fragments were then transformed into S. pneumoniae and select with Columbia blood agar with 1 µg/ml tetracycline.

Gene deletions

Erythromycin resistant marker (eryR) was used as the selection marker for all the deletion mutants in this study. To delete *lafB* gene (SPV_0960), upstream and downstream of *lafB* coding regions were amplified with OVL4184/OVL4185 and OVL4180/4181 oligo pairs, respectively. The DNA sequence containing the coding sequence and ribosome binding site of eryR was amplified with OVL2933/OVL2934. The three amplified upstream, downstream and eryR fragments were then assembled by Golden Gate Assembly using BsaI as the restriction enzyme. The product was then transformed into S. pneumoniae and selected with Columbia blood agar with 0.5 µg/ml erythromycin. Deletion of cps locus was performed with the same strategy, and the oligo pairs to amplify the upstream and downstream homologous arms were OVL4610/OVL4611 and OVL4608/OVL4609, respectively.

Complementary strains

The complementary strain of *lafB* was made by introducing an ectopic *lafB* driven by its native promoter on ZIP locus of S. pneumoniae.⁸⁴ The upstream locus of ZIP locus with a spectinomycin resistant marker was amplified from pPEPZ⁸⁴ with OVL3252/OVL3253, and the downstream of the ZIP locus was amplified from pPEPZ with OVL3254/OVL3255. The two acquired fragments were then digested with BsmBI. The promoter and coding region of LafB was amplified from the genomic DNA of S. pneumoniae D39V with OVL4441 and OVL4442, followed by BsaI digestion. Then the three digested fragments were ligated. The ligation product was then transformed into S. pneumoniae and the transformants were selected with Columbia blood agar with 100 µg/ml spectinomycin. As a control for the *lafB* complementary strain, the empty pPEPZ was transformed into S. pneumoniae and the selection was performed in the same way.

GFP fusion strains

In this study, a C-terminal and N-terminal GFP fused LafB were constructed both driven by an IPTG-inducible promoter at the ZIP locus. To make the N-terminal version of the fusion, we first amplified 3 fragments. The first was a DNA fragment with the upstream of ZIP, spectinomycin resistant marker specR, Plac promoter, and the msfGFP. The fragment was amplified from pASR108⁸⁴ with oligos OVL1841/OVL5845. The second fragment was the LafB coding region without start codon and was amplified from genomic DNA of S. pneumoniae D39V with oligos OVL5846/OVL5847. The third fragment was the downstream of ZIP locus and was amplified from pASR108 with oligos OVL5851/OVL3255. The three fragments were digested with BsmBI, followed by ligation. The ligation product was transformed into S. pneumoniae and 100 µg/ml spectinomycin was used for selection. The C-terminal version of fusion was constructed in a similar way, whereas the oligo pairs OVL1841/OVL5849, OVL5850/OVL5853, OVL5851/OVL3255 were used for amplification of the three fragments.

IPTG-inducible HiBiT-tagged *lafB* mutant

Both N- and C-terminal HiBiT-tagged LafB were constructed at the CIL locus⁸⁴ under an IPTG-inducible promoter. Firstly, an IPTG-inducible *lafB* was inserted at the CIL locus, and the produced strain was VL4018. To construct VL4018, three fragments were acquired. The first fragment containing upstream of CIL locus and kanamycin resistant marker was amplified from pASR105 with OVL3318/OVL3371, followed by Nhel digestion. The second fragment with Plac-*lafB* was amplified from VL4007 with OVL1754/OVL1225, followed by Xhol and Nhel double digestion. The third fragment with downstream of the CIL locus was amplified from pASR105 with oligos OVL925/OVL3321, followed by Xhol digestion. The three digested fragment was then ligated and transformed into S. pneumonie VL333 to construct VL4018.

To make the HiBiT-tagged *lafB* on N terminal, two fragments were amplified, *BsmBI* digested and ligated, followed by transformation and selection with 150 µg/ml kanamycin. The first fragment containing upstream of CIL locus, kanamycin resistant marker, and IPTG inducible promoter was amplified from VL4018 with OVL6063/OVL3318. The second fragment containing hibit-tagged *lafB* and downstream of CIL locus was amplified from VL4018 with OVL6064/OVL3321. The C terminal HiBiT-tagged LafB was constructed in a similar way, but OVL3318/6065 and OVL6066/OVL3321 were used to amplify the two fragments from VL4018, respectively.

Construction of the CRISPRi libraries

The plasmids with the sgRNA pool were purified with an *E. coli* library (Addgene #170432), and then transformed into different S. pneumoniae strains as well described previously.⁸⁴

Construction of luciferase reporter strain (VL2255)

This strain was constructed based on VL2212.³⁴ The sgRNA targeting *luc* gene was cloned into vector pPEPZ-sgRNAclone (Addgene #141090) as described previously,⁸⁴ and the produced plasmid was named as pPEPZ-sgRNAluc. The oligos for annealing of the spacer sequence of sgRNAluc were OVL1020/OVL1021. The pPEPZ-sgRNAluc was then transformed into VL2212 and selected with 100 µg/ml spectinomycin on Columbia agar plates.

Construct the CPD-tagged *LafB* protein expression plasmid

The *lafB* gene was cloned into the plasmid pLIBT7_A_CPDHisOld for tagging and expression. The *lafB* gene fragment was amplified from genomic DNA of S. pneumoniae D39V with two oligos OVL4449/OVL4450, while the backbone of plasmid pLIBT7_A_CPDHisOld was amplified with two oligos "STM121_Bsalins_rev_notag" and "CPDHis_NEW_forward_with_pIDC_overhang". The two fragments were then assembled by Golden Gate Assembly with BsaI as the restriction enzyme. The product was transformed into chemically competent *E. coli* DH5α, and the transformants were selected on LB agar with 100 µg/ml ampicillin.
The successfully cloned plasmid "pLIBT7_A_lafB-CPDHisOld" was confirmed by sanger sequencing. For induction of protein expression, the plasmid was transformed into *E. coli* BL21 freshly.

### Construction of CRISPRi mutants targeting *cozE* and *divIB*

The sgRNAs targeting *cozE* and *divIB* were cloned into vector pPEPZ-sgRNAclone (Addgene #141090) as described previously.⁸⁴ Oligos with the spacer sequences targeting *cozE* and *divIB* were OXL812/OXL813 and OXL814/OXL815, respectively. The pPEPZ-sgRNA vectors with the sgRNAs were then transformed into VL4017 and selected with 100 µg/ml spectinomycin on Columbia agar plates.

### Antisera production in rabbit used for western blotting

100 µg/injection of protein was used to raise antibodies in one rabbit, and in total 4 injections were performed on day 0, 7, 10, 19, following the Speedy 28-Day program of Eurogentec. The produced antiserum from the immunized rabbit was shown to be immunogenic against, and specific for pneumococcal LafB by Western blotting.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Data analyses were performed with GraphPad Prism (v8.0) and R (v4.1.1). Data shown in plots are averages of at least 3 replicates with SEM. For animal infection assays, at least 5 mice were used for each group, and differences were determined using the Mann-Whitney U test for comparing two groups, Kruskal-Wallis test with Dunn's post-analysis for comparing multiple groups. p values were stated in the figure legends.

## ADDITIONAL RESOURCES

No additional resources.

## DATA AND MATERIALS AVAILABILITY

All data are available in the main text or the supplementary materials. CRISPRi-seq data are available at NCBI Sequence Read Archive: PRJNA895037.