# When the Host Encounters the Cell Wall and Vice Versa

Kelvin Kho, Thimoro Cheng, Nienke Buddelmeijer, Ivo Gomperts Boneca

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Kelvin Kho, Thimoro Cheng, Nienke Buddelmeijer, Ivo Gomperts Boneca. When the Host Encounters the Cell Wall and Vice Versa. Annual Review of Microbiology, 2024, 78 (1), pp.233-253. 10.1146/annurev-micro-041522-094053. pasteur-05124620

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ANNUAL
REVIEWS

*Annual Review of Microbiology*

# When the Host Encounters the Cell Wall and Vice Versa

Kelvin Kho, Thimoro Cheng, Nienke Buddelmeijer, and Ivo G. Boneca

Institut Pasteur, Université Paris Cité, Paris, CNRS UMR 6047 Integrative and Molecular Microbiology, INSERM U1306 Host-Microbe Interactions and Pathophysiology, Unit Biology and Genetics of the Bacterial Cell Wall, France; email: bonecai@pasteur.fr

Annu. Rev. Microbiol. 2024. 78:12.1–12.21

The *Annual Review of Microbiology* is online at
micro.annualreviews.org

https://doi.org/10.1146/annurev-micro-041522-094053

Copyright © 2024 by the author(s).
All rights reserved

## Keywords

peptidoglycan, cell wall, antimicrobials, host responses, innate immunity

## Abstract

Peptidoglycan (PGN) and associated surface structures such as secondary polymers and capsules have a central role in the physiology of bacteria. The exoskeletal PGN heteropolymer is the major determinant of cell shape and allows bacteria to withstand cytoplasmic turgor pressure. Thus, its assembly, expansion, and remodeling during cell growth and division need to be highly regulated to avoid compromising cell survival. Similarly, regulation of the assembly impacts bacterial cell shape; distinct shapes enhance fitness in different ecological niches, such as the host. Because bacterial cell wall components, in particular PGN, are exposed to the environment and unique to bacteria, these have been coopted during evolution by eukaryotes to detect bacteria. Furthermore, the essential role of the cell wall in bacterial survival has made PGN an important signaling molecule in the dialog between host and microbes and a target of many host responses. Millions of years of coevolution have resulted in a pivotal role for PGN fragments in shaping host physiology and in establishing a long-lasting symbiosis between microbes and the host. Thus, perturbations of this dialog can lead to pathologies such as chronic inflammatory diseases. Similarly, pathogens have devised sophisticated strategies to manipulate the system to enhance their survival and growth.

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## Contents

<table><tr><td>1. INTRODUCTION</td><td>12.2</td></tr><tr><td>2. DYNAMICS OF CELL WALL GROWTH</td><td>12.2</td></tr><tr><td>3. TURNOVER, RECYCLING, AND SHEDDING OF PEPTIDOGLYCAN</td><td>12.5</td></tr><tr><td>4. CELL SHAPE AND PATHOGENICITY</td><td>12.6</td></tr><tr><td>5. MECHANISMS OF RESISTANCE TO HOST RESPONSES, ANTIMICROBIAL PEPTIDES, AND LECTINS</td><td>12.8</td></tr><tr><td>5.1. Modification of Effector Targets</td><td>12.8</td></tr><tr><td>5.2. Modulation of Surface Properties</td><td>12.9</td></tr><tr><td>5.3. Disabling Host Effectors</td><td>12.10</td></tr><tr><td>6. NOVEL WAYS TO TARGET THE CELL WALL BY BLOCKING PATHOGENESIS</td><td>12.10</td></tr><tr><td>6.1. Targeting Mechanisms of Resistance</td><td>12.10</td></tr><tr><td>6.2. Targeting Mechanisms of Peptidoglycan Shedding and Recycling</td><td>12.10</td></tr><tr><td>7. MECHANISMS OF HOST RECOGNITION: INNATE IMMUNE RECEPTORS AND EFFECTORS TARGETING THE CELL WALL</td><td>12.11</td></tr><tr><td>8. CELL WALL-MEDIATED HOST EFFECTS MODULATING HOST RESPONSES</td><td>12.12</td></tr></table>

## 1. INTRODUCTION

Peptidoglycan (PGN), the major component of the bacterial cell wall, is composed of chains of sugars cross-linked by short peptides and is a central structure in the bacterial cell envelope, which provides resistance to turgor pressure and shape to the bacterial cell. To maintain cell envelope integrity, PGN is physically connected to other cell components in the cell envelope, including outer membrane proteins, lipoproteins, and teichoic or mycolic acids. The sugar entities, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), are conserved. However, stem peptide variations; chemical modification of sugars; and attachment of capsules, teichoic acids, and arabinogalactan polymers occur in different species (135). These structural diversities play an important role in recognition by host cells and binding to innate immune receptors and thus affect immune responses to both commensal and pathogenic bacteria. Hence, understanding how PGN and the cell wall assembly are regulated has a direct impact on how bacteria interact with their environment, particularly with the host. In this review, we introduce the latest research on PGN: its biosynthesis, its shedding, how it regulates cell shape, how it impacts bacteria-host interactions of both commensal and pathogens, and how it interacts with the host and influences its response.

## 2. DYNAMICS OF CELL WALL GROWTH

PGN is a dynamic structure that allows bacterial cells to grow, divide, and undergo shape transitions under various environmental conditions. Incorporation of new building blocks is catalyzed by enzymes that possess transglycosylase activity to connect sugar moieties and transpeptidase activity to assure peptide cross-link formation. While most synthetic enzymes are classified as penicillin-binding proteins (PBPs) through historical studies on inhibition by β-lactam antibiotics, a role for the so-called SEDS (shape, elongation, division and sporulation) proteins in transglycosylation has been reported (90, 125). To guarantee correct synthesis of the cell wall, both transglycosylase

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<footnote n="12">12.2 Kho et al.</footnote>
and transpeptidase activities are required. The SEDS proteins depend on class B PBP counterparts for complementary transpeptidase activity, while class A PBPs are bifunctional enzymes. A second class of enzymes, collectively termed hydrolases and lyases, have PGN-degrading activities (Figure 1). Their action creates space for the incorporation of new material into the existing layer, recycling of PGN fragments, and shedding of PGN particles into the environment. Glucosaminidases and muramidases are hydrolytic enzymes cleaving the β-1,4 linkage between GlcNAc-MurNAc and MurNAc-GlcNAc, respectively. Lytic transglycosylases (LTs) also digest the MurNAc-GlcNAc sugar bond, forming 1,6-anhydro-MurNAc. Digestion of peptide bonds is catalyzed by endopeptidases (within the stem peptide and cross-links), carboxypeptidases (which trim the stem peptide sequentially from the C terminus), and amidases (between MurNAc and the stem peptide). Both synthesis and degrading activities need to be coordinated in space and time for essential cellular processes such as growth, division, differentiation, and adaptation to occur. Recent studies have addressed the question of how PGN biogenesis is regulated. Here we highlight the most recent findings not covered by recent excellent reviews [see also the review by Brogan & Rudner (19)]. Several examples have been described in which protein-protein interactions between enzymes and regulators play a key role. The regulation of amidases by partners located in the outer membrane and an energy-providing complex in the cytoplasmic membrane is prototypical. Various species of *Enterobacteriaceae* harbor multiple amidases. The amidases AmiA and AmiB in *Escherichia coli* are regulated through interaction with the M23-like peptidase EnvC, which connects to FtsX, while AmiC is regulated by outer membrane lipoprotein NlpD. Recent work provides insight into the mechanism of the FtsEX-EnvC-AmiB complex of *Pseudomonas aeruginosa* through the release of regulatory helices in both EnvC and AmiB upon ATP binding by FtsE (143). All three amidases are regulated by ActS under different environmental conditions (95); ActS also activates a MepS-dependent pathway (see below) (25). AmiC, the single-cell separation amidase in *Neisseriaceae*, is also regulated by NlpD (82) and releases PGN-derived peptides that activate NOD1 (23). RipA, an essential cell separation D,L-endopeptidase in *Corynebacterium glutamicum*, is controlled by autoinhibition via a coiled-coil domain and by interaction with a cytoplasmic membrane complex other than FtsEX (44). Recent findings suggest that the inactive LytM-type peptidase DipM interacts with a multitude of lytic enzymes to coordinate PGN turnover for daughter cell separation in *Caulobacter crescentus* (59). LytH in *Staphylococcus aureus* is a membrane-anchored amidase that cleaves peptides from non-cross-linked glycan strands. In the absence of LytH, cells become large and display a cell division defect. LytH is active only in the presence of its membrane-bound regulator ActH (101), whereby its binding stabilizes Zn²⁺ in the active site (101). In *Clostridium difficile*, CwlD is also regulated by allosteric interaction with GerS, facilitating Zn²⁺ binding and activity (2). These examples demonstrate that regulation of PGN turnover is established by events taking place in both the outer membrane and cytoplasmic membrane through stretching of coiled-coil domains and movements of occluding α-helices to allow for coordinated action in PGN biogenesis.

A second level of regulation is through proteolytic processing. Three endopeptidases, MepS, MepM, and MepH, of *E. coli* are controlled via the periplasmic protease Prc upon targeting by NlpI (62). Prc also cleaves the LT MltG (57), providing a central role in PGN turnover. Scaffolding of hydrolases by NlpI likely involves additional PGN synthesis enzymes and their regulators, as demonstrated through multiple protein interactions (9). Processing of Atl by secreted proteases in *S. aureus* yields the mature glucosaminidase GlcA and AmiA separately (100). The glucosaminidase AtlA of *Enterococcus faecalis* is processed at the N terminus (120). What the effect of proteolytic processing is on enzyme function and how the activity of proteases is regulated require more investigation.

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<figure><img src="image_6.png" /><figcaption>12.4 Kho et al.</figcaption></figure>

(Caption appears on following page)

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<caption>**Figure 1** (*Figure appears on preceding page*)</caption>

A schematic diagram of the pathways involved in bacterial PGN remodeling, of PGN sensing and targeting by the host, and how bacteria adapt to resist these host responses. (a,b) Both gram-negative diderms (a) and gram-positive monoderms (b) have a PGN layer that, in conjunction with cytoskeletal proteins, determines the shape of the bacteria. Although PGN architecture varies with species, they express specific and overlapping enzymes for different steps in PGN cleavage. (c) The muropeptides liberated are then transported into the bacterial cytoplasm via transporters like AmpG for recycling, and they can also be released into the milieu either directly or via outer membrane vesicles. (d) Host cells then sense these muropeptides through surface receptors like C-type lectins and the Toll-like receptor 2-CD14 complex. (e) In parallel, liberated muropeptides can be taken up by the host via the transporters SLC15A1/2/3/4 and SLC46A2/3. (f) The uptake of these PGN muropeptides triggers the cytoplasmic receptors NOD1 and NOD2, which recognize specific muropeptide architecture and inhibit mitochondrial hexokinase via the GlcNAc moiety; these then lead to the expression of proinflammatory genes and subsequently disease. (g) Secreted host factors such as lysozyme and PGLYRP2 degrade PGN to lyse bacteria and release immunostimulatory molecules; although PGLYRP1/3/4 have PGN-binding domains, their activities are unclear. (h) To negate these host effectors, bacteria respond by modulating their surface properties. One way is by tailoring PGN to prevent recognition by host receptors and enzymes. (i) Moreover, the charges on the bacterial cell surface can be tuned to repel these effectors either by decorating LPSs in diderms and teichoic acids in monoderms or by adjusting the lengths of these polymers through, e.g., controlling the activity of TagF in monoderms. (j) Surface charges are also modulated by aminoacylating phosphatidylglycerol via MprF. (k) In addition, host effectors can be directly neutralized such as by inhibiting lysozyme via the surface-exposed lipoprotein SliC. (l) SpA, which is both localized by teichoic acids and secreted, can neutralize IgGs by binding to their Fc region and can prevent antibody development by adaptive immunity B cells. (m) To mitigate cell envelope stresses, various two-component pathways like the WalKR system become activated to adapt the expression of cell wall remodeling proteins. As many of these aforementioned proteins are important mechanisms of resistance to host responses, they serve as antibiotic targets of interest. Abbreviations: GlcNAc, N-acetylglucosamine; LPS, lipopolysaccharide; MurNAc, N-acetylmuramic acid; PGN, peptidoglycan; SpA, staphylococcal protein A.

Third, cellular localization of enzymes allows control of activity at specific sites in the cell. Particularly important is mid-cell localization of protein complexes composed of PGN-degrading and PGN-synthesizing enzymes for correct cell division. AmiB and AmiC are recruited to the division site independently of their respective regulators. AmiB and NlpD depend on the cell division protein FtsN for localization (103). Interaction of AmiA with the LytM domain of EnvC, which is connected to the FtsEX energy-providing complex (28) and is connected to the cell division protein FtsA (37), displaces the autoinhibitory helix in AmiA, leading to activation of AmiA (27). The regulation of amidases in *E. coli* depends on environmental cues (95), suggesting that niche-occupying species such as *Helicobacter pylori* (24) and *Neisseriaceae* regulate their sole amidase through a single control mechanism. The cell wall hydrolase LytB is regulated by the protein kinase StkP and through interaction with wall teichoic acids (WTAs) in the final cell division step of *Streptococcus pneumoniae* (89).

While many findings are reported on PGN dynamics during cell division, less is known about regulation of PGN biogenesis during cell elongation. A large protein complex central to NlpI includes a transglycosylase and its regulator, carboxypeptidases, and endopeptidases (9). A well-studied, fourth system of regulation is transcriptional control of hydrolases required for cell growth by the two-component regulatory system WalKR in *Firmicutes* (36). CwlO and LytE are the only endolytic enzymes (out of 42) that are essential for cell growth in *Bacillus subtilis* (140). WalR controls the expression of *cwlO* and *lytE* as well as that of *iseA*, which encodes a LytE inhibitor, and *pdaC*, which encodes a PGN deacetylase (36). Recent studies on *B. subtilis* L-forms suggest that CwlO and LytE have specific functions acting via class A PBPs and RodA, respectively (68). The activity of the WalK component is stimulated by the two accessory membrane proteins YycH and YycI in *S. aureus* through phosphorylation (45).

### 3. TURNOVER, RECYCLING, AND SHEDDING OF PEPTIDOGLYCAN

PGN-degrading activity results in release of fragments (as muropeptides) that are reused in novel synthesis or released either in the form of soluble molecules or as part of outer membrane vesicles (OMVs) and, as such, signal the host immune system [Figure 1; see also review by Kwan

<figure><img src="image_8.png" /></figure>
& Qiao (77)]. Muropeptides are transported back to the cytoplasm through a permease (AmpG) in the form of GlcNAc-anhMurNAc-peptides or GlcNAc-anhMurNAc, which requires LT and amidase activities in the periplasm. These PGN fragments are further processed by amidases, carboxypeptidases, and glucosaminidases. Fragments are then sensed by the transcription factor AmpR, which regulates *ampC* expression. *ampC* encodes β-lactamase. Expression of *ampC* and production of β-lactamase lead to resistance to β-lactam antibiotics. The mechanism of regulation is not well understood, but it depends on the relative amount of muropeptides present. An alternative pathway is signaling by PGN fragments via two-component systems. Posttranslational modification of receptors by phosphorylation or acylation upon PGN fragment sensing leads to activation of gene expression by the regulatory component. Moreover, released tripeptides can be incorporated into new PGN, or it can be further processed into amino acids. How these processes are regulated is an open question.

PGN fragments released into the environment serve as signaling molecules for interbacterial communication and stimulation of the host innate immune system. Recent work demonstrated the important role of membrane vesicles in the release and uptake of molecules, including PGN, by host cells [see review by Toyofuku (129)]. Different types of vesicles are produced by bacteria, depending on the cell envelope composition and mechanisms of formation. In general, membrane blebbing and cell lysis triggered by endolysins are two mechanisms of vesiculation in which the cross-link between the membrane and PGN is weakened. OMV formation is increased in the absence of the PGN synthase activator LpoA in *Haemophilus influenzae* (61) and in the absence of the PGN-bound lipoprotein Pal in *Bordetella pertussis* (34). Release of Pal in OMVs is observed in *E. coli* upon exposure to ampicillin (92). Several other examples show the loss of the physical interactions between the outer membrane and PGN under stress conditions leading to OMV formation (33, 102, 118). A role for NlpI in OMV formation was described in *E. coli*, in which a double mutant of phospholipid transport components *mlaA* and *nlpI* led to hypervesiculation (99). Similarly, *nlpI* was a hit in a screen for mutants defective in toxin secretion via OMVs in *Salmonella enterica* Typhi (96).

Monoderm bacteria also produce membrane vesicles through blebbing and through a cell lysis mechanism termed bubbling cell death (129). Stress conditions and endolysins are involved, but extensive knowledge on membrane vesicle biogenesis is lacking.

## 4. CELL SHAPE AND PATHOGENICITY

PGN is a major determinant of bacterial cell shape; the growth dynamics and spatial arrangement of PGN influence morphology. These processes are orchestrated by two main cellular machineries known as the elongasome and the divisome. Thus, it is not surprising to see the recent growing interest in the morphology and true diversity of bacteria. Bacteria come in many forms varying from rodlike and spherical to spiral, cube, and starlike, and the motivation for such research goes beyond curiosity in basic microbiology. Bacteria's ability to establish, maintain, and pass on their unique shapes to subsequent generations suggests its importance in bacterial lifestyle. Bacterial cell shape has so far been associated with survival in environmental niches, immune evasion, and pathogenicity (64).

Accordingly, different species are known to possess unique cytoskeletal components for fine-tuning their special shapes (Figure 1). For example, rather than adopting a classical straight-rod morphology, species such as *Vibrio cholerae*, the causative agent of cholera epidemic, take on a curved-rod morphology. *V. cholerae* possesses a specialized component known as CrvA that can polymerize into filaments at the inner face of cell curvature. CrvA has been suggested to function as a periskeletal element that contributes to asymmetrical growth rates between the inner and outer face of *V. cholerae* in a density-dependent manner. The straight rod-shaped ΔcrvA mutant

<figure><img src="image_9.png" /></figure>
was found to be defective in motility in a viscous environment and to poorly colonize in both mouse and rabbit models (10, 86).

Other excellent illustrations of the link between bacterial cell shape and pathogenicity are found in helical-shaped bacteria, including *H. pylori* and *Leptospira* species. The helical shape of *H. pylori* aids in its movement in a viscous environment in a corkscrew-like manner and was thus suggested to have a crucial role in *H. pylori* survival in the host gastric mucosa (87). Several cell shape determinants, including PGN hydrolases and bactofilins, are known, and inactivation of any of these genes leads to cell shape defects that render the species incapable of colonizing the host (17, 123, 124). Likewise, the helical-shaped *Leptospira* have long been known for their active motility and ability to bore into mucous membranes of host tissues. These virulence factors are implicated in their morphology. The helical shape of *Leptospira* is generally believed to play a central role in motility and infection. While a direct implication has not been demonstrated in vivo, mutants lacking the cytoskeletal component LbbD adopt a compressed helical morphology with motility defects, displaying decreased ability to move through soft agar medium, and are significantly lower in velocity compared to wild type (60). Since motility and surface penetration play crucial roles in the infection cycle of *Leptospira*, this study strongly suggests an important role for the *Leptospira* cell shape in pathogenicity, warranting a follow-up study in vivo. Additionally, while the helical shape of *Leptospira* is dictated by its PGN—and not by the flagella, as is the case for its close relative *Borrelia burgdorferi*—its two periplasmic flagella have been demonstrated to bend the ends of *Leptospira* cells into long pitch spirals (S) or flat half-circle hook shapes (H) (105). These ends can adopt multiple global configurations to confer different modes of motility crucial to *Leptospira* migration and colonization inside hosts (126, 127).

One remarkable discovery in bacterial morphology is its plasticity. There has been growing evidence that bacterial cell shape is less static than previously thought and can undergo changes via evolution or dynamic processes under the influence of environmental conditions (146). A change of cell shape from rods to cocci in *Neisseria meningitidis*, the causative agent of the deadly meningococcal disease, was selected for through evolution to allow for better colonization of its niche, the nasopharynx. This morphological transition involves at least a deletion of a single gene implicated in the transition from cell elongation to cell division, *yacF* (132). These cocci have a smaller surface-to-volume ratio than do rods; this property has been hypothesized to make *N. meningitidis* more resistant against host immune aggression and to have dispersed type IV pili that could reinforce adhesion to the nasopharyngeal mucosa.

Bacteria also dynamically undergo shape transitions from one shape to another within a span of several generations. *Campylobacter jejuni*, a spiral curved species capable of inhabiting the gut of humans and many other animals, took the shape of curved rods after 24 h of incubation in vitro and eventually became round or spherical after 48 h (21). Krogfelt et al. (73) followed the growth of *E. coli* BJ4 in mice in vivo and found that this strain differentiated into two populations distinguishable by their colony size, growth rate, and cell morphology despite having the same serotype, biotype, and plasmid profiles. The significance of this dynamically adaptive morphogenesis is clearly seen in infection-related filamentation. Such filamentation is a stress-induced mechanism that is central to the infection cycle of many pathogens, including uropathogenic *E. coli* (UPEC), which is the leading cause of urinary tract infections (64, 131). UPEC undergoes multiple morphological transformations during its entire infection cycle from rods to cocci, then back to rods and some filaments before eventually transforming back to classical rods (3, 63, 64). Upon invasion into the superficial umbrella cells of the bladder, UPEC develops complex intracellular bacterial communities (IBCs). The first morphological transformation from rods to cocci, during which the average length is reduced from 3 µm to 1 µm, has been designated the hallmark of the middle-stage IBCs. While the significance of this length reduction is

<figure><img src="image_10.png" /></figure>
unclear, it has been speculated that this specific shape contributes to the global arrangement of the highly dense and organized bacterial communities observed. Filamentation of UPEC occurs in late-stage IBCs and is mediated by the cell division inhibitor SulA in response to DNA damage. This morphological change is essential to the UPEC pathogenic cascade but is dispensable in Toll-like receptor 4 (TLR4)-knockout mice, indicating its role in protecting against the host immune response, perhaps phagocytosis, as well as against antibiotics that act on cell division (65). Nonetheless, this filamentation is transient, and these bacteria eventually revert to classical rods through division mostly near the ends of the elongated cells. The transient filamentation has been proposed to aid in UPEC dispersal from the IBCs and host cell into the extracellular environment. Fascinatingly, the division taking place in this stage is independent of FtsZ, the classical divisome initiator, and is instead mediated by DamX, a member of the SPOR domain family of proteins that bind PGN (119). Ironically, DamX has also been suggested to contribute to blockage of cell division during filamentation since mutants of its gene abrogated the filament phenotype (69). Altogether, it is speculated that SulA-mediated filamentation and DamX-mediated filamentation in UPEC are triggered by two distinct pathways but work in concert to promote pathogenesis.

## 5. MECHANISMS OF RESISTANCE TO HOST RESPONSES, ANTIMICROBIAL PEPTIDES, AND LECTINS

Host organisms harbor a variety of mechanisms to control bacterial populations, whether in their microbiome or during an infection. Strategies employed include the production of enzymes like lysozyme as well as antimicrobial peptides (AMPs). These molecules share a common feature whereby they attack the bacterial cell wall to weaken PGN or destabilize bacterial membranes, ultimately resulting in lysis. In response to these pressures, bacteria have adapted diverse mechanisms to survive and persist within their niche in hosts. Many of these basic mechanisms have been reviewed elsewhere, and here we focus on the most recent advances in the field (1, 8).

### 5.1. Modification of Effector Targets

Enzymes targeting the bacterial PGN are relatively specific with regard to the moieties that they recognize (Figure 1). The standard PGN building block is GlcNAc-MurNAc-pentapeptide, but many bacteria have adapted their PGN to avoid targeting by hydrolytic enzymes, as was thoroughly reviewed previously (144). The most common modifications identified (along with the involved enzymes) are O-deacetylation (the O-acetyl esterase Ape) and O-acetylation of MurNAc (the O-acetyltransferases OatA, Pat/Pac, Adr), which occurs in numerous species (122). In addition, N-deacetylation of GlcNAc (PgdA, PdaV) can be largely found in opportunistic monoderms (56, 136). Rarer modifications include N-glycosylation of MurNAc (NamH) (109), which occurs in Mycobacteria; N-deacetylation of MurNAc (PdaA, PdaB, and PdaC) in *Bacillus* species (40, 41, 71); and O-acetylation of GlcNAc (OatB) in *Lactiplantibacillus plantarum* (14). These modifications are important for pathogenicity; in *Legionella pneumophila*, loss of PdgA causes the bacteria to mislocalize to lysosomes and sensitizes it to lysozymes (16). Moreover, the regulation and effects of PGN modification can be complex. For example, *C. difficile* encodes both *pgdA* and *pdaV*: The former is constitutively expressed, while the latter is inducible upon lysozyme action (30). Only the deletion of both enzymes sensitizes *C. difficile*. Yet, this is not linked to any marked decrease in virulence (67). As a gram-positive pathogen, *E. faecalis* alters its cell wall in multiple ways, including D-alanylating teichoic acids (discussed further below), O-acetylating PGN MurNAc, and deacetylating PGN GlcNAc. However, it is the latter that confers resistance to the enzymatic action of lysozyme and lysozyme's cationic antimicrobial activity (13).

<figure><img src="image_11.png" /></figure>
The basic pentameric stem peptide of PGN, L-Ala-*iso*-D-Glu-(L-Lys or M-DAP)-D-Ala-D-Ala, is also commonly modified, but it mainly counteracts antibiotic action. The most common resistance mechanism is swapping the terminal D-Ala-D-Ala with a D-Ala-D-Lac to resist vancomycin. The third amino acid and the variety of transpeptidases produced by bacteria also dictate the degree of cross-linking and subsequently the structural integrity of PGN when faced with different effectors. Intriguingly, recent work has identified a modification of the fourth stem peptide amino acid, D-alanine, to alaninol in *S. typhimurium*, and this was shown to attenuate proinflammatory signaling (53). Furthermore, *B. burgdorferi* uniquely produces PGN-containing GlcNAc-GlcNAc, which resists lysozyme action and potentially induces noncanonical PGN innate immunity responses (35).

## 5.2. Modulation of Surface Properties

AMPs exert their effect by virtue of the charges they carry to insert themselves into bacterial membranes to form destabilizing pores and subsequently kill bacteria. To counteract these effects, bacteria have adapted different mechanisms to prevent these molecules from reaching their membranes, mainly by producing a repulsive force on their envelopes (Figure 1). The activity of the multiple peptide resistance factor (*mprF*) has long been established as a key component in modulating membrane charges by aminoacylating phosphatidylglycerol with recently uncovered roles in global lipid homeostasis (108).

Modification of non-PGN surface saccharides can also modulate surface properties to resist cationic AMPs. The GlcNAc deacetylase of *Streptococcus pyogenes*, also known as group A *Streptococcus* (GAS), putatively deacetylates poly-GlcNAc (7). This confers fitness advantages in infection models via lysozyme resistance and modifies surface charges to confer resistance to the cationic AMP cecropin B. Moreover, GAS expresses the GlcNAc deacetylase PplD (113). This enzyme deacetylates not PGN, but the linkage GlcNAc on the surface Lancefield group A carbohydrate. This putatively increases the net positive charge on the cell surface to resist the cationic human group IIA secretory phospholipase A2 (hGIIA), which is necessary for membrane attack complex formation and bacteria killing by neutrophils (52).

Diderm bacteria express lipopolysaccharides (LPSs) on their surface, conferring the ability of surface charges to resist environmental insults. Various LPS modifications, as reviewed elsewhere, are important for bacteria to resist these insults (85). These modifications include adding palmitate, aminoarabinose, galactosamine, and phosphoethanolamine to decrease the net negative charge of the core lipid A. Moreover, LPSs are also acylated and deacylated to control membrane permeability and attenuate immune responses. Recently, it was shown that *Shigella flexneri* resists host antimicrobial fatty acids by increasing its O-antigen lengths (5).

In contrast, the monoderm cell wall is covered by polyol phosphate glycopolymers referred to as teichoic acid. To mask these net negative charges, teichoic acids are modified by D-alanylation. The loss of this modification is associated with fitness defects and with increases in susceptibility to AMPs and lysozyme. Additionally, teichoic acid D-alanylation can be found in tandem with PGN modification (4). Teichoic acids are also glycosylated, but to a lesser extent. Although loss of glycosylation does not result in fitness defects, glycosylated WTAs are recognized by serum antibodies and pattern recognition molecules like mannose-binding lectins (76). Remarkably, *S. aureus* changes its WTA glycosylation patterns upon infection in mice (94), indicating an advantage in host colonization or immune evasion. In response to lysozyme stress, the metalloprotease Eep in *E. faecalis* is essential to activate the stress response cascade. This includes the upregulation of GlcNAc deacetylation, but the main mode of lysozyme resistance is dependent on the WTA polymerase TagF homolog in the enterococcal polysaccharide antigen locus (112). Lipoteichoic acids (LTAs) are teichoic acid anchored in the membrane by a diglucosyl-diacylglycerol (DGDAG)

<figure><img src="image_12.png" /></figure>
linkage unit, whereby they are also glycosylated, but the implications of these modifications are
still unclear (70). However, LTA lengths are modulated by the availability of DGDAG (55), as
implicated by *mprF* studies. LTAs impact bacterial physiology and potentially antimicrobial re-
sistance, drawing parallels with LPS (54). A recent study also identified populations of *E. faecalis*
that upregulate DGDAG when penetrating epithelial cells with marked increase in resistance to
cationic antimicrobial peptides (107).

**5.3. Disabling Host Effectors**

The most direct way for bacteria to persist in their host is by subjugating immune system ef-
fectors (Figure 1). Lysozyme is the most ubiquitous effector, and bacteria have evolved various
basic strategies to counter it, as reviewed elsewhere (106). Pathogens like *Neisseria gonorrhoeae* pro-
duce the surface-exposed lipoprotein SliC, which directly binds to and inactivates lysozyme (150).
*B. pertussis* is also capable of binding to and disabling AMPs by virtue of the *Bordetella* polysac-
charide, which is displayed both on the cell surface and as a secreted factor (42). This allows the
bacteria to infect the respiratory tract, and surprisingly, expression of this polysaccharide in *E. coli*
could also confer the same pathology. During bacterial infections, the classical complement acti-
vation pathway of the immune system kicks off by direct binding of antibodies to bacterial surface
antigens. However, *S. aureus* directly abrogates this by expressing surface protein A (39), which
is in turn localized on the bacterial surface by D-alanylation of LTA (149). This protein prevents
IgG hexamer formation by sequestering the Fcγ domain and, in addition, cross-links IgM Fab
domains, preventing antibody development (31).

# 6. NOVEL WAYS TO TARGET THE CELL WALL
## BY BLOCKING PATHOGENESIS

**6.1. Targeting Mechanisms of Resistance**

Cell wall modifications both are essential for resisting host responses and are heavily implicated
in growth fitness, and thus these pathways are targets of interest for antimicrobial development
(Figure 1). MurNAc O-deacetylation of PGN is a widespread phenomenon implicated in
pathogen fitness and host colonization (51, 133), with evidence of cooperation with PGN-
remodeling enzymes (139). Hence, there have been multiple efforts to identify inhibitors of the
enzyme Ape. These resulted in the identification of purpurin from a high-throughput screen
(104) and potent rationally designed aldehyde-based inhibitors (137). Efforts have also been di-
rected to identify inhibitors of the N-deacetylase PgdA. Virtual screening and computational
docking have yielded several promising inhibitors of PgdA with activity in vitro (22). Elsewhere,
high-throughput screens for O-acetylase inhibitors identified benzothiazolyl-pyrazolo-pyridine
as inhibiting PatB from N. gonorrhoeae as well as coumarin-based inhibitors of PatB and OatA
in S. aureus (20). There are no reported efforts to discover inhibitors of MurNAc N-glycosylase,
MurNAc N-deacetylase, and GlcNAc O-acetylase, mainly due to the limited number of bacteria
that express these modifications. As the bacterial capsule is also implicated in pathogenesis, the
potency of several hit compounds targeting the S. pneumoniae uridine diphosphate glucose py-
rophosphorylase (29), a key step in capsule biosynthesis, was evaluated. Despite a lack of growth
inhibition in vitro, these compounds attenuated pneumonococcal virulence in a *Galleria mellonella*
infection model.

**6.2. Targeting Mechanisms of Peptidoglycan Shedding and Recycling**

As discussed above, cell wall hydrolases must be tightly regulated to maintain cell wall integrity and
to prevent lysis. As such, these components serve as attractive targets and an alternative to direct

<figure><img src="image_13.png" /></figure>
targeting of PGN synthesis machineries (Figure 1). For example, NlpI serves as a scaffold for PGN hydrolases (9), and it directs the proteolysis of all D,D-endopeptidases in *E. coli* (62). The class of NlpC/P60 cysteine peptidases is also a target of interest (49). In bacteria, these endopeptidases are important for degrading the PGN stem peptide for PGN expansion and recycling. Similarly, in the gram-positive *S. aureus*, disruptions to the WalKR regulatory system simultaneously confers resistance to most cell wall antibiotics yet sensitizes the bacteria to tunicamycin due to a collapse in the PGN recycling pathway (128). Interestingly, the PGN recycling pathway is also implicated in the resuscitation of spores and dormant bacteria (98, 117); inhibition of this pathway would allow for the targeting of these recalcitrant forms of bacteria.

There is active interest in identifying specific inhibitors of enzymes in the PGN recycling path-
way. Specifically, LTs as antimicrobial targets is a field of interest (88). Bulgecin A was identified
as a specific inhibitor of LTs that has synergy with β-lactam antibiotics. Since then, a structure-
guided study based on GlcNAc derivatives identified several lead compounds for C.jejuni LT (75),
whereas thionine, a peptidic lysozyme inhibitor from plants, was discovered to be a weak inhibitor
of E. coli Slt35 (91). Other enzymes in the recycling pathway include the inner membrane permease
AmpG. AmpG imports GlcNAc-1,6-anhydromuropeptides, which are then processed by NagZ,
a β-N-acetylglucosaminidase. Genetic deletions of either AmpG or NagZ largely phenocopied
each other, and small-molecule inhibitors of NagZ were previously reported and further devel-
oped (121, 145). Further downstream in the PGN recycling pathway is the GlcNAc-6-phosphate
deacetylase NagA; pyrimirhodomyrtone was recently identified as an inhibitor of S. aureus NagA
with efficacy in mice skin infection models (147). Dithiazoline was identified as an inhibitor of
L,D-carboxypeptidase A (LdcA) (12). LdcA is required to process the recycled stem tetrapeptides
to tripeptides prior to reincorporation of these into the de novo biosynthesis of PGN precursors.
Although LdcA is essential only during the stationary phase in E. coli, more recently it was shown
to inhibit N. gonorrhoeae in all growth phases (81), suggesting that it could be used as a target in
certain species.

7. MECHANISMS OF HOST RECOGNITION: INNATE IMMUNE RECEPTORS AND EFFECTORS TARGETING THE CELL WALL

Historically, PGN and cell wall are terms that have been used interchangeably in the literature, even though they are not synonymous. PGN is indeed the major component of the bacterial cell wall, but cell walls also include secondary polymers such as teichoic acids, capsules, covalently anchored proteins, cell wall-associated proteins such as S-layers, and lipoproteins. Thus, the roles of the cell wall and PGN in host-microbe interactions can be overlapping but not equivalent, as cell wall biological effects can be mediated by cell wall components other than PGN. Because of the unique nature of bacterial PGN, it was coopted during evolution as a biomarker of bacterial origin that is targeted both by effector molecules and by signaling receptors central to the host-microbe dialog.

The first-ever-described effector molecule, lysozyme, which hydrolyzes PGN, leads to bacterial lysis and release of soluble PGN fragments (114) (Figure 1). Other effector molecules include PGN-binding proteins that function both as hydrolytic enzymes [PGLYRP-2, also known as serum amidase (38)] and as AMPs [PGLYRP1, -3, and -4 (38)]. C-type lectins have also been described as effector molecules. In general, these bind to PGN through recognition of the GlcNAc residue of PGN. Many of these activate after binding the lectin-complement pathway [e.g., ficolins (83)], while others, such as RegIII C-type lectins, act as AMPs (79).

A major advancement in understanding the host-microbe dialog came with the discovery of pattern recognition receptors (PRRs), signaling receptors that sense PGN. CD14 was the first-ever receptor shown to bind PGN and to signal through the NF-κB pathway (50). But since these

<figure><img src="image_14.png" /></figure>
discoveries in 1990s, few additional studies have dissected the role of CD14 in PGN signaling. However, CD14 is not essential for PGN sensing. Interestingly, CD14 has been described as a coreceptor to Toll-like receptor 2 (TLR2). In the 1990s, several studies claimed that TLR2 was the PRR that sensed PGN (115), a discovery that is still controversial today. What is clear from the literature is that TLR2 senses lipoproteins, a common contaminant of PGN preparations (130). While biochemical studies have shown that TLR2 can bind soluble PGN fragments, their ability to signal remains a question in the field (6).

The field made a significant advancement with the discovery of the PRRs Nod1 and Nod2 (of the NLR family) in the early 2000s as the bona fide receptors of PGN fragments. Nod1 and Nod2 are multimodular cytosolic PRRs composed of an effector CARD domain, a NOD-activating domain, and a leucin-rich ligand-binding domain. Both sense PGN fragments, i.e., *m*-DAP-containing muramyltripeptide and muramyldipeptide (MDP), through their respective leucin-rich domains (47). Nod1 and Nod2 have attracted much attention since their discovery due to their central role in immune homeostasis and their role in multiple chronic inflammatory diseases (e.g., Crohn's disease, Blau syndrome, asthma). Other PRRs, such as NLRP1 and NLRP3, calreticulin, and the serotonin receptors, have been suggested to sense PGN fragments (for a recent review, see Reference 11). However, these have been poorly studied and remain to be fully characterized. More recently, PGN-derived GlcNAc was shown to bind to hexokinase-1 and to promote NLRP3 activation (141). Independent of their individual contributions to PGN sensing, all these receptors have in common the ability to sense PGN fragments rather than the PGN polymer, and thus the implication is that sensing of PGN requires prior processing of the PGN layer by both endogenous PGN hydrolases and host enzymes such as lysozyme. Thus, understanding the regulation of cell wall growth and turnover is central to appreciating the impact that these released PGN fragments can have on the host.

## 8. CELL WALL-MEDIATED HOST EFFECTS MODULATING HOST RESPONSES

The first detailed characterization of biological effects of PGN fragments was performed in the 1970s and 1980s, when muropeptides were discovered to have major adjuvant and immunomodulatory activities (93). Soon after, many other effects, such as enhanced protection against infectious challenges through the stimulation of immune cells (32), modulation of sleep (74), and regulation of bone mass (110), were described. In parallel, PGN fragments were shown to be deleterious; for example, they induced apoptosis of ciliated tracheal epithelial cells (111). This last point is reminiscent of the role of PGN fragments in the development of the light organ of the Hawaiian squid (72). Hence, these PGN fragments can have opposing consequences. This is a clear trademark of the physiological effects of PGN fragments on host physiology that range from beneficial immunomodulatory effects in anticancer therapy to deleterious chronic inflammatory consequences in arthritis.

The yin and yang effect of PGN fragments on the host was partially forgotten when the Nod1 and Nod2 PRRs were discovered. Their cytosolic localization and role as the first line of defense in the gastrointestinal tract led to the hypothesis that Nod1 and Nod2 were innate immune sentinels in the sterile environment of the cell, allowing the host to distinguish between beneficial microflora and pathogens. Accordingly, other innate immune receptors such as the membrane-exposed TLRs are mostly silent in epithelial cells (through, e.g., downregulation, absence of coreceptors, special regulation restricted to the basolateral side of the epithelial barrier). This view changed in 2008, when it was shown that development and maturation of isolated lymphoid follicles—major immune structures of the gut that produce and secrete IgAs, among other

<figure><img src="image_15.png" /></figure>
effectors—were regulated by the released PGN fragments from the commensal gram-negative intestinal flora (18). Soon after, Clarke and colleagues provided evidence that neutrophil priming in the bone marrow required the systemic dissemination of PGN fragments generated by the intestinal flora (26). Since then, the roles of PGN fragments in shaping host physiology, particularly in regulating host immune responses, have regained major attention from the scientific community. Several studies have provided convincing evidence for an important stimulatory role of PGN fragments in anticancer therapy. Chemotherapy and immune checkpoint blocker immunotherapy have been shown to depend on effects mediated by the microbiota (134). Certain microbial communities seem more able than others to enhance the immune response. Part of this beneficial effect is mediated through Nod2, implicating its ligands as well. The underlying mechanisms were recently dissected. Enhanced release of Nod2 ligands by enterococcal strains requires the production of the D,L-endopeptidase SagA (48). Transfer of SagA activity to a non-SagA-producing related enterococcal strain led to genetically modified *Enterococcus* capable of reproducing the phenotypes of the original donor enterococcal strain (48). This is reminiscent of studies showing that certain probiotic strains with anti-inflammatory properties in models of chronic intestinal inflammatory disease also seem to produce more Nod2 ligands (84). This finding was supported by a recent study that mined metagenomes for genes associated with protection against intestinal bowel diseases such as Crohn's disease (46). These metagenomes were enriched in genes encoding D,L-endopeptidase activities capable of generating MDP (46). In addition to having effects on the immune system, PGN fragments, through Nod2 signaling, support intestinal stem cell survival and regeneration (97) against oxidative stress-mediated cell death. Similarly, Nod2 sensing of microbiota-derived PGN by the epithelium improves growth during undernourishment (116). However, most of these studies focused on the role of PGN fragments in restoring and maintaining homeostasis.

Besides the beneficial effects of PGN fragments in restoring homeostasis, there is also growing evidence for other physiological effects of PGN fragments on host tissues. For example, a recent study showed that microbiota-derived MDP modulates appetite and body temperature through Nod2 in GABAergic neurons of the hypothalamus (43). Similarly, Nod1 ligands released by the microbiota through the action of lysozyme regulate insulin trafficking in the pancreas (148) and the adrenomedullary response in chromaffin cells (142). These studies clearly support an emerging role of PGN fragments in regulating many biological processes of the body.

However, under certain conditions, PGN fragments have been associated with diseases. The best-described pathology involving PGN is arthritis. Septic arthritis due to bacterial infections has directly implicated PGN in the inflammatory process such as during borreliosis (66). Moreover, PGN has been implicated as a driver of even noninfectious rheumatoid arthritis. Neutralization of circulating PGN fragments with anti-MDP antibodies ameliorates disease (58). Similarly, such neutralization also prevents neurodegenerative diseases such as multiple sclerosis. Indeed, PGN seems to be a driver of several chronic inflammatory diseases, including chronic brain inflammation (78) and psoriasis (15).

However, a major caveat of these studies is the tools used to dissect the role of PGN in the host. Many of these studies used knockout mice for Nod1 and Nod2 but failed to demonstrate that their ligands reached the different distant organs and tissues involved in the process. Attempts to detect PGN fragments in these parts either were based on radiotracking experiments (11) or used methods to estimate the total amount of PGN in a given sample by analyzing the total muscular acid content (80). The best direct evidence for biologically active PGN fragments comes from studies using antibody-mediated detection of PGN in host fluids (58). The future of the field of PGN-host dialog depends on the ability to detect and analyze the chemical nature of the PGN fragments circulating in the host, as these could become biomarkers of disease. More recent

<figure><img src="image_16.png" /></figure>
analyses used high-resolution mass spectrometry to characterize the diversity of PGN fragments
of a complex microbiome (138), which was hence termed the peptidoglycome. The ability to track
PGN fragments in the host will be instrumental to understanding their biology, as many effects
depend on their chemical nature, how they are modified, and how they are eventually eliminated
by the host.

In conclusion, this review presents an integrated view of the current state of the art on the bacterial cell wall, in particular in the context of the host-microbe interactions, and discusses how we can explore this knowledge both to improve our therapeutic strategies against pathogenic bacteria and to modulate the host response toward these same bacteria.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We would like to thank the members of the Boneca laboratory. The Boneca laboratory was sup-
ported by the following programs: Investissement d'Avenir program, Laboratoire d'Excellence
"Integrative Biology of Emerging Infectious Diseases" (ANR-10-LABX-62-IBEID); Investisse-
ment d'Avenir program, INCEPTION (ANR-16-CONV-0005); Investissement d'Avenir pro-
gram, RHU Torino Lumière (ANR-16-RHUS-0008); the French National Research Agency
(ANR-16-CE15-0021); FRM Grant Programme d'Urgence (DBF20160635726); and R&D
grants from Danone and MEIJI. K. Kho was supported by the Pasteur-Roux-Cantarini Postdoc-
toral Fellowship from the Institut Pasteur. T. Cheng was supported by a PhD fellowship from the
Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases."

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