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# The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence

Devon Kavanaugh, Constance Porrini, Rozenn Dervyn, Nalini Ramarao

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Devon Kavanaugh, Constance Porrini, Rozenn Dervyn, Nalini Ramarao. The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence. PLoS ONE, 2022, 17 (1), pp.1-13. 10.1371/journal.pone.0259386. http://dx.doi.org/10.1371/journal.pone.0259386

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RESEARCH ARTICLE

# The pathogenic biomarker alcohol dehydrogenase protein is involved in *Bacillus cereus* virulence and survival against host innate defence

Devon W. Kavanaugh<sup>D</sup>, Constance Porrini<sup>D</sup>, Rozenn Dervyn, Nalini Ramarao<sup>D</sup>*

Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

* nalini.ramarao@inrae.fr

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## Abstract

*Bacillus cereus* is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. *B. cereus* is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both *in vitro* and *in vivo*. Our results demonstrate that the *adhB* gene plays a significant role in resistance to nitric oxide and oxidative stress *in vitro*, as well as its pathogenic ability with regards to *in vivo* toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

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OPEN ACCESS

**Citation:** Kavanaugh DW, Porrini C, Dervyn R, Ramarao N (2022) The pathogenic biomarker alcohol dehydrogenase protein is involved in *Bacillus cereus* virulence and survival against host innate defence. PLoS ONE 17(1): e0259386.
https://doi.org/10.1371/journal.pone.0259386

**Editor:** Vijay Tripathi, Sam Higginbottom University of Agriculture, Technology and Sciences, INDIA

**Received:** May 25, 2021

**Accepted:** October 18, 2021

**Published:** January 4, 2022

**Copyright:** © 2022 Kavanaugh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the manuscript.

**Funding:** The author(s) received no specific funding for this work.

**Competing interests:** The authors have declared that no competing interests exist.

## Introduction

*Bacillus cereus* is an ubiquitous spore forming human pathogen. It is present in soil, foods, almost all surfaces in hospital settings, and human skin. It is the second leading cause of collective foodborne outbreaks in France after *Staphylococcus aureus* and the third in Europe [1-3]. *B. cereus* was associated with 155 outbreaks, 1,636 illnesses and 44 hospitalizations in Europe in 2019 according to reports by 27-member states. *B. cereus* can induce two types of gastrointestinal diseases, leading to emetic or diarrhoeal syndromes. *B. cereus* can also cause severe systemic infections, especially in immunocompromised patients leading to patient death in approximately 10% of cases [4-9]. However, some *B. cereus* strains can cause severe and even fatal infections in healthy people [10]. The pathogenic potential of *B. cereus* is thus extremely variable, with some strains being harmless and others lethal [11].
*B. cereus* produces toxins such as Hbl, Nhe, and CytK that induce cell toxicity [12–14]. In addition, other factors such as HlyII, InhA1, CwpFM or Mfd have been implicated in *B. cereus* resistance against the host immune system [15–21]. These toxins provide an indication of the strain toxicity potential [13, 22–24]. However, these factors do not allow the discrimination of strains according to their pathogenicity. Indeed, several studies have shown that the Nhe production by hazardous strains is variable and that non-pathogenic strains can also produce it in large quantities [1, 24]. Moreover, these toxins do not appear to be suitable markers for strains causing non-gastrointestinal infections [22].

*B. cereus* strains that induced severe gastrointestinal or non-gastrointestinal disorders do not carry neither *hbl*, *ces*, *hlyII*, *cytK1* nor *cytK2* genes and did not produce the Nhe protein, implying that other still unknown factors were responsible for their pathogenicity [1, 11].

Accordingly, we have recently analyzed a large strain collection comparing strains that induced an infection (intestinal or otherwise) with non-pathogenic strains [11, 25]. The large strain screening allowed to identify a combination of four as yet undescribed biomarkers, wherein their presence/absence allows an accurate identification of clinical *B. cereus* strains [26]. Three of these genes are located on the bacterial chromosome, and the fourth one is located on a large plasmid in a region that could be defined as a novel pathogenicity island for *B. cereus* [27]. These findings constitute a huge step in the understanding of the *B. cereus* pathogenic potential and complexity and may provide tools to better assess the risks associated with *B. cereus* contamination. Among these genes, *adhB*, was identified as a leading candidate [26]. This *adhB* gene encodes an alcohol dehydrogenase-like protein (ADH). This family of enzymes is involved in oxidation-reduction biological process. ADH are involved in metabolic and physiological processes in a variety of organisms, including fermentative metabolism [28], the oxidation of alcohols as carbon and energy sources [29], protection against anaerobic stress [30], and maintenance of the intracellular redox balance [31].

In this study, the *adhB* gene was knocked out to better elucidate its function during *B. cereus* virulence. Our results demonstrate that *adhB* plays a significant role in resistance to nitric oxide (NO) and oxidative stress *in vitro*, as well as its pathogenic ability with regards to *in vivo* infection and toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

# Materials and methods

## Bacterial strains

This study includes 35 *B. cereus* strains isolated from human patients following systemic or local infections and 21 non-pathogenic strains (Table 1). The 35 strains of the clinical collection were isolated from patient samples (biopsy, blood culture, etc) from nine French voluntary hospitals between 2008 and 2014. The samples and information were collected for a previous study and were treated anonymously and thus not subjected to personal consent [22]. The non-pathogenic strains have been isolated from food, where no infection was reported in humans. They were further tested in cell and animal models and did not induce any pathologies [23, 25]. We have previously shown a correlation between cytotoxicity and virulence [11]. Nevertheless, although these strains had previously been shown to be weakly cytotoxic to human cells and to have reduced virulence in an insect infection model, this does not rule out their potential ability to produce symptoms in specific vulnerable populations (i.e. the elderly, immunocompromised, or premature/new-born babies).
<table><caption>Table 1. Characteristics of non-pathogenic (A) and clinical (B) strains.</caption><thead><tr><th>Non-pathogenic strains</th><th>Source</th><th>adhB</th></tr></thead><tbody><tr><td>INRA-PF_S09</td><td>Milk protein</td><td>0</td></tr><tr><td>I13_S10</td><td>Cooked rice</td><td>1</td></tr><tr><td>INRA-5_S11</td><td>Pasteurized zucchini puree</td><td>0</td></tr><tr><td>INRA-C64_S12</td><td>Pasteurized vegetables</td><td>0</td></tr><tr><td>ADRIA-I3_S13</td><td>Cooked foods</td><td>0</td></tr><tr><td>INRA-BN_S36</td><td>Vegetable</td><td>1</td></tr><tr><td>INRA-PA_S37</td><td>Milk protein</td><td>0</td></tr><tr><td>INRA-A3_S38</td><td>Starch</td><td>1</td></tr><tr><td>I23_S39</td><td>Cooked apple</td><td>0</td></tr><tr><td>SB_S40</td><td>Soil from a vegetable field</td><td>0</td></tr><tr><td>I11_S41</td><td>Cooked food</td><td>1</td></tr><tr><td>INRA-C1_S42</td><td>Pasteurized vegetables</td><td>0</td></tr><tr><td>INRA-C46_S43</td><td>Pasteurized vegetables</td><td>0</td></tr><tr><td>INRA-SL_S44</td><td>Soil</td><td>0</td></tr><tr><td>INRA-SO_S45</td><td>Soil</td><td>0</td></tr><tr><td>INRA-BC_S47</td><td>Vegetable</td><td>1</td></tr><tr><td>I2_S48</td><td>Dried fruit</td><td>0</td></tr><tr><td>INRA-BL_S49</td><td>Vegetable</td><td>0</td></tr><tr><td>ADRIA I21_S50</td><td>Cooked foods</td><td>0</td></tr><tr><td>INRA-SV_S51</td><td>Soil</td><td>0</td></tr><tr><td>WSBC 10204_S52</td><td>Pasteurized milk</td><td>0</td></tr></tbody></table>

B

<table><thead><tr><th>Clinical strains</th><th>Age of patients</th><th>Type of sampling</th><th>Symptoms</th><th>Outcomes</th><th>adhB</th></tr></thead><tbody><tr><td>09CEB13BAC_S6</td><td>Premature newborn</td><td>Blood culture</td><td>Brain abscess</td><td>Recovery</td><td>1</td></tr><tr><td>09CEB14BAC_S7</td><td>Premature newborn</td><td>Blood culture</td><td>Bacteremia</td><td>Recovery</td><td>1</td></tr><tr><td>09CEB33BAC_S8</td><td>Newborn</td><td>Axilla-later feces</td><td>Skin infection</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB31BAC_S14</td><td>Premature newborn</td><td>Blood culture</td><td>Organ failure and pulmonary and cerebral abscesses</td><td>Death</td><td>1</td></tr><tr><td>13CEB06BAC_S15</td><td>86</td><td>Blood culture from catheter</td><td>Heart failure, ventilator-associated pneumonia, ischemic stroke</td><td>Recovery</td><td>1</td></tr><tr><td>09CEB11BAC_S16</td><td>Premature newborn</td><td>Blood culture</td><td>Meningitis, infection in the liver, both lungs</td><td>Death</td><td>1</td></tr><tr><td>09CEB16BAC_S17</td><td>Newborn</td><td>Umbilical</td><td>Local colonization</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB30BAC_S18</td><td>Premature newborn</td><td>Blood culture</td><td>Sepsis</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB40BAC_S20</td><td>63</td><td>Blood culture</td><td>Bacteremia and central venous catheter-linked infection</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB46BAC_S21</td><td>61</td><td>Blood culture</td><td>Sepsis (patient with an acute myeloid leukemia)</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB47BAC_S22</td><td>43</td><td>Blood culture</td><td>Bacteremia</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB51BAC_S23</td><td>60</td><td>Blood culture</td><td>Sternum abscess, absent fever</td><td>Sequela of osteitis</td><td>1</td></tr><tr><td>13CEB01BAC_S24</td><td>31</td><td>Prosthesis from tibia</td><td>No clinical sign of infection</td><td>Recovery</td><td>1</td></tr><tr><td>09CEB12BAC_S53</td><td>Premature newborn</td><td>Cerebrospinal fluid</td><td>Meningitis, infection in the liver, both lungs</td><td>Death</td><td>1</td></tr><tr><td>09CEB34BAC_S59</td><td>Premature newborn</td><td>Stomach-tube feeding</td><td>Premature birth</td><td>Recovery</td><td>1</td></tr><tr><td>09CEB36BAC_S61</td><td>Premature newborn</td><td>Central venous catheter</td><td>Bacteremia</td><td>Recovery</td><td>1</td></tr></tbody></table>

(Continued)
<table><caption>**Table 1.** (Continued)</caption><tr><td>12CEB34BAC_S64</td><td>80</td><td>Thoracentesis</td><td>Pulmonary infection</td><td>not known</td><td>1</td></tr><tr><td>12CEB37BAC_S90</td><td>30</td><td>Blood culture</td><td>Endocarditis</td><td>Death</td><td>1</td></tr><tr><td>12CEB38BAC_S91</td><td>65</td><td>Blood culture</td><td>Sepsis</td><td>Death</td><td>1</td></tr><tr><td>12CEB39BAC_S92</td><td>54</td><td>Blood culture</td><td>Sepsis</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB42BAC_S94</td><td>63</td><td>Blood culture</td><td>Bacteremia and central venous catheter-linked infection</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB43BAC_S95</td><td>63</td><td>Blood culture</td><td>Bacteremia and central venous catheter-linked infection</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB44BAC_S96</td><td>34</td><td>Blood culture</td><td>Bacteremia</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB45BAC_S97</td><td>newborn</td><td>Blood culture</td><td>Kidneys and urinary infections</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB48BAC_S98</td><td>66</td><td>Blood culture</td><td>Bacteremia (patient with a colorectal cancer)</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB49BAC_S99</td><td>24</td><td>Blood culture+ skin infection</td><td>Sepsis and aplastic anemia caused by drugs</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB50BAC_S100</td><td>77</td><td>Blood culture</td><td>Bacteremia (patient with breast cancer)</td><td>Recovery</td><td>1</td></tr><tr><td>12CEB52BAC_S101</td><td>40</td><td>Blood culture</td><td>Bacteremia (immunocompromised patient)</td><td>Recovery</td><td>0</td></tr><tr><td>13CEB03BAC_S102</td><td>76</td><td>Blood culture</td><td>Community acquired pneumonia</td><td>Recovery</td><td>1</td></tr><tr><td>13CEB07BAC_S105</td><td>24</td><td>Blood culture</td><td>Abdominal pain, shivering, vomiting, fever, diarrhea</td><td>Recovery</td><td>1</td></tr><tr><td>13CEB09BAC_S106</td><td>85</td><td>Liver abscess</td><td>Sepsis, hepatitis c and liver abscess, abdominal pain, diarrhea</td><td>Recovery</td><td>1</td></tr><tr><td>13CEB30BAC_S107</td><td>not known</td><td>Blood culture</td><td>Nausea, abdominal pain and vomiting</td><td>not known</td><td>1</td></tr><tr><td>14CEB16BAC_S114</td><td>Premature newborn</td><td>Blood culture from peripheral veins</td><td>Septic shock, multiple organ failure, pulmonary and cerebral abscesses</td><td>Death</td><td>1</td></tr><tr><td>14CEB17BAC_S115</td><td>Premature newborn</td><td>Bronchial aspiration (lung)</td><td>Septic shock and pneumonia<br>pulmonary necrotic abscesses, recurrent pneumothorax</td><td>Death</td><td>1</td></tr><tr><td>14SBCL987_S116</td><td>not known</td><td>Biopsy (kidney)</td><td>Vomiting and diarrhea</td><td>Death</td><td>1</td></tr></table>

The absence (0) or presence (1) of the *adhB* gene was detected by PCR.

https://doi.org/10.1371/journal.pone.0259386.t001

## *adhB* gene detection by PCR

For all the strains, a single colony was picked, resuspended in 100 µL Tris-EDTA NaCl buffer (TEN) and incubated at 98°C for 10 min. After centrifugation to pellet cell debris, 1 µl of supernatant was used as DNA matrix. The PCR mixture for gene detection contained 1 µl DNA matrix, 0.5 µM primer (forward: TTATTATCTATTCTTTTCGTGTGATGC, and reverse CTATTTGTAGCAGAACATTCRAAACC), 10 µL DreamTaq Green PCR Master Mix (2X) (Thermo Scientific) in a final volume of 20 µL. Thermal cycling was carried out in a Mastercycler® nexus (Eppendorf) with the following program: a start cycle of 3 min at 98°C, followed by 30 cycles of 20 s at 98°C, 30 s at 55°C, and 1 min at 72°C, and a final extension time of 10 min at 72°C. PCR fragment sizes were revealed on 1.5% agarose gels containing Midori Green, and visualised by a UV imaging device as previously described [26].

## *adhB* mutant generation

The Bt407 Cry- with the reference genome *Bacillus thuringiensis* Bt407: NC_018877.1 was used as a model for *B. cereus* and was renamed Bc 407.

Knock-out of the *adhB* gene (WP_000438843) was accomplished by double-cross over gene substitution by use of the pMAD vector [32]. Briefly, using the available sequencing information of the Bc407 strain, 600 bp regions upstream and downstream of the identified gene of interest were synthesized surrounding a tetracycline-resistance cassette by the GeneCust company (Boynes, France). The upstream nucleotide coordinates used are 2,575,680 to 2,576,279, and the downstream nucleotide coordinates are 2,577,204 to 2,577,802. The synthesized region was then ligated into the pMAD vector. This vector was further transformed by heat shock into chemically competent NEB-10 beta cells. The plasmid was then extracted and transformed
into *E. coli* strain ET to facilitate de-methylation of the plasmid, increasing subsequent trans-
formation into *B. cereus* *Bc407* as previously described [16]. Resulting colonies were then sub-
jected to temperature stress at 40°C to force the incorporation of the resistance cassette leading
to the stable knock-out of the *adhB* gene, which was verified by PCR with oligonucleotide
sequences flanking the cloned region. The mutation was stable and sequencing revealed that
the mutation occurred at the corrected place and did not affect the flanking regions. The
resulting strain was designated as *ΔadhB*.

Wild type and mutant strains were streaked onto BHI agar from 20% glycerol stocks to
obtain isolated colonies. Colonies were inoculated into BHI broth and grown at 37°C, 200 rpm
until mid to late-exponential phase for phenotypic analysis. Cultures in mid-exponential phase
were used for microscopy to determine cellular morphology. For growth assays, stocks were
inoculated into BHI broth and followed by sampling for CFU/ml at regular intervals.

Nitric oxide (NO) stress survival

*B. cereus* *Bc407* and the Δ*adhB* mutant were grown to late-exponential phase. Cultures were harvested and diluted 1:1000 in RPMI (Gibco Glutamax, Fisher Scientific, Illkirch Cedex, France) and further grown at 37°C without agitation with differing doses of the NO donor, NOC-5 (3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (Calbiochem, Sigma-Aldrich, Saint-Louis, MO, USA). NOC-5 was dissolved in NaOH 0.01 M and used at the following concentrations: 0, 15.6, 25, 31.25, 50, 62.5, 100, 125, 250, 500 µM. After 1 h, bacteria were agitated to avoid sedimentation and the survival rate was quantified after 4 h by plating serial dilutions on LB agar plates. Data are pooled from two to four independent experiments and presented as % survival = (NO-treated/Buffer-treated) × 100.

Oxidative stress survival

Oxidative stress-resistance was determined as previously described [33]. Briefly, wild-type and Δ*adhB* mutant strains were grown and 2 h post-inoculation, 500 µl of each culture was added to 100 µl of either sterile water or hydrogen peroxide at final concentrations of 2 mM or 10 mM. Treated (2 mM or 10 mM H₂O₂) and control (H₂O₂) cultures were incubated for 10 min at 37°C and then serially diluted in phosphate-buffered saline (PBS) and plated on BHI to stop the reaction and count CFU/ml. Data are pooled from two independent experiments and presented as % survival = (H₂O₂-treated/H₂O₂-treated) × 100.

Insect infection trial

*B. cereus* *Bc407* and the Δ*adhB* mutant were grown to exponential phase. Cultures were har-
vested and serially diluted 1:4 in peptone water prior to injection. 10 to 20 last instar *Galleria
mellonella* larvae were used following a 24 h fast as previously described [34, 35]. 10 µl of bacte-
rial preparations at various doses were injected between the second and third body segment
from the rear of the insect. Injected insects were incubated at 37°C for 24 h, following which
survival was assessed. Peptone water was injected as negative control. Data are pooled from
three independent experiments and presented as % survival = (injected with strain/injected
with water) x 100.

**Protein bioinformatic analysis**

The protein sequences of the ADH protein (WP_000438843) was analysed with Pfam to find
functional domains. E-values are based on searching the Pfam-A family against UniProtKB
2018_04 using HMM search.
<figure><img src="image_5.png" /></figure>

<table><thead><tr><th rowspan="2">Domain</th><th rowspan="2">Start</th><th rowspan="2">End</th><th colspan="2">Gathering threshold (bits)</th><th colspan="2">Score (bits)</th><th colspan="2">E-value</th></tr><tr><th>Sequence</th><th>Domain</th><th>Sequence</th><th>Domain</th><th>Sequence</th><th>Domain</th></tr></thead><tbody><tr><td>ADH_N</td><td>26</td><td>91</td><td>21.10</td><td>21.10</td><td>33.90</td><td>25.10</td><td>0.00015</td><td>0.079</td></tr><tr><td>ADH_zinc_N</td><td>147</td><td>265</td><td>31.80</td><td>31.80</td><td>33.30</td><td>32.60</td><td>0.00025</td><td>0.00042</td></tr></tbody></table>

**Fig 1. Structural domains of AdhB.** The AdhB protein of *B. cereus* is composed of a catalytic domain with an inserted zinc-binding domain (green box) and a co-factor-binding domain at its C terminus (red box). E-values are based on searching the Pfam-A family against UniProtKB 2018_04 using hmmsearch.

https://doi.org/10.1371/journal.pone.0259386.g001

## Statistical analysis

Statistical analysis was performed with GraphPad Prism version 7. Insect survival curves were assessed by non-linear regression, constraining the bottom to 0.

Bacterial survival rate following stresses were also analysed by non-linear regression, and the statistical differences were calculated with a Wilcoxon test between the conditions with or without stress.

## Results

### *adhB* as a marker of clinical *B. cereus* strains

The presence/absence of the *adhB* gene was assessed by PCR on a collection of strains of varying pathogenic potential: 21 non-pathogenic strains and 35 clinical strains (Table 1). *adhB* was present in 34/35 (97%) clinical isolates, whereas it was present in 5 of 21 (24%) non-pathogenic isolates. We thus hypothesised that *adhB* may be a new and important virulence factor of *B. cereus*.

The amino acid sequences of the Bc407 gene WP_000438843 coding for a protein of the AdhB family was analysed using the Uniprot database (Fig 1). This enzyme of 308 amino acids belongs to the zinc-containing alcohol dehydrogenase family. The software identified two domains, with the catalytic domain of the alcohol dehydrogenase containing an inserted zinc-binding domain. This domain has a GroES-like structure [36, 37]. The co-factor-binding domain of the enzyme is located proximal to the C-terminus. Structural studies indicate that it forms a classical motif called Rossman fold that reversibly binds NAD(H) as a co-factor [38, 39].

### Growth characteristics and morphology

*B. cereus* Bc407 and the Δ*adhB* mutant were grown in BHI medium at 37°C, 200 rpm and bacterial growth was followed by measuring the OD₆₀₀, and CFU/mL determined by serial dilution and plating (Fig 2A and 2B). The two strains presented similar rates of growth with no significant differences in growth curves. The strains were observed under the microscope and bacterial morphology shows that the two strains are similar in cellular shape and size, with the *adhB* mutant often making longer chains of cells (6–8 cells) (Fig 2C and 2D).
<figure><img src="image_6.png" /><figcaption>**Fig 2. Bacterial growth curves and cellular morphology.** Bacterial growth was determined by calculating CFU/mL (A) or following optical density at 600 nm (B) for the wildtype *B. cereus* Bc407 (▲; solid line) and the Δ*adhB* mutant (●; dashed line) strains. Representative bacterial morphology of Bt407 WT (C) and *adhB* mutant (D) are viewed at 100x magnification. The scale bar represents a length of 10 µm. All graphs represent one representative experiment out of three biological replicates.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0259386.g002

## Nitric oxide (NO) and oxidative stress resistance

To assess the role of AdhB in the resistance to the host immune system response, *B. cereus* Bc407 and Δ*adhB* strains were incubated with the NO donor to test their resistance against NO stress (Fig 3). Several doses of NO were assessed and the dose inhibiting 50% of bacterial growth (IC50) was calculated. The IC50 of *B. cereus* wild type (WT) strain is approximately 4 times higher than that of the mutant (193 vs 45 µM of NO) and the survival rate of the mutant is lower at each concentration of NO tested. Thus, the mutant *adhB* is more sensitive to nitric oxide than the wild type strain.

Then, oxidative stress resistance of *B. cereus* Bc407 WT and Δ*adhB* strains was determined after exposure to 2 mM or 10 mM H₂O₂ for 10 min at 37°C (Fig 4). Wildtype Bc407 demonstrated increased resistance at both concentrations, with survival percentage being 14-fold higher at 2 mM, and 20-fold higher at 10 mM.

## Insect model of *B. cereus* toxicity

The role of AdhB in the pathogenicity of *B. cereus* was assessed in an insect model of infection. *B. cereus* Bc407 and Δ*adhB* mutant strains were injected at various doses into *Galleria mellonella* larvae (Fig 5). At 24 h post-injection, survival of the insects was assessed. Insects infected with the Δ*adhB* mutant strain demonstrated higher rates of survival in relation to the wildtype strain, demonstrating a reduced virulence of the mutant strain. Further, statistical analysis of the survival curves reveals a significant difference in the LD50 values between the strains: 4.2 10³ CFU/injection for the wildtype and 1.5 10⁴ CFU/injection for the Δ*adhB* mutant. HillSlope determined the curves to be distinct at 99.94% probability.
<figure><img src="image_7.png" /><figcaption>**Fig 3. NO sensitivity.** The wild type and ΔadhB mutant strains were cultured and incubated for 4 h in the presence of different concentrations of NO donors. Bacterial survival was quantified by plating and bacterial resistance to NO was measured and normalized with respect to the control condition, without NO. Data points correspond to the mean ± SEM of the values obtained from 2 to 4 biological replicates. The calculation of the IC50 of NO was performed using Graphpad.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0259386.g003

Discussion

Alcohol dehydrogenase (ADH) is an enzyme involved in oxidation-reduction biological process. It catalyses the reversible oxidation of alcohols and induces the formation of their corresponding acetaldehyde or ketone with the reduction of NAD (Fig 6). This class of enzyme typically has a broad spectrum of action [40, 41]. Here we characterized AdhB as a protein involved in *B. cereus* resistance to nitric and oxidative stresses, two major components of the host immune system, and in its pathogenicity.

Currently three types of alcohol dehydrogenases are known, that differ structurally and catalytically: Zinc-containing 'long-chain' alcohol dehydrogenases, 'short-chain' alcohol dehydrogenases, and iron-containing alcohol dehydrogenases [42, 43]. The AdhB (WP_000438843) protein in *B. cereus* is a zinc-containing ADH. These enzymes are typically dimeric or tetrameric proteins, which require two atoms of zinc per subunit to be functional, however, catalytic activity is maintained in the presence of a single zinc atom. The zinc atoms interact with either cysteine or histidine residues; the catalytic zinc being coordinated by two cysteines and one histidine. Zinc-containing ADH's are found in bacteria, mammals, plants, and fungi. Normally, there is more than one isozyme per species (e.g. humans possess at least six isozymes and yeast have three). Consistently, we identified three Zinc-containing ADH's in the Bc407 strain (WP_000438843, WP_000649129.1, WP_000645827.1). These three isozymes share common structures with two identified domains (not shown). The first is the catalytic domain that might contain an inserted zinc-binding domain. This domain has a GroES-like structure; a name derived from the superfamily of proteins with a GroES fold. Proteins with a GroES fold structure have a highly conserved hydrophobic core and a glycyl-aspartate dipeptide,
<figure><img src="image_8.png" /><figcaption>**Fig 4. H₂O₂ sensitivity.** The wild-type and ΔadhB mutant strains were grown and subsequently exposed to either 2 mM or 10 mM of hydrogen peroxide for 10 min at 37°C. Bacterial survival was assessed by plating and normalized against buffer-treated controls. Data points correspond to the mean ± SEM of the values obtained from 2 biological replicates.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0259386.g004

<figure><img src="image_9.png" /><figcaption>**Fig 5. Insect infection.** Bacterial virulence was determined as *Galleria mellonella* survival percentage following injection with varying CFU/mL of wild type (triangles, black line) or ΔadhB (circles, dashed line) mutant strains. Survival was measured as live insects following 24 h post-injection. Calculation of the LD50 was done using Graphpad software.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0259386.g005
<figure><img src="image_10.png" /><figcaption>**Fig 6.** Reaction catalyzed by an alcohol dehydrogenase. The alcohol dehydrogenase catalyzes the oxidation of alcohol into their corresponding aldehyde (primary alcohol) or ketone (secondary alcohol) with the reduction of NAD+.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0259386.g006

which is thought to maintain the fold. The second is the domain that binds its cofactor NAD
owing to its motif denoted as a Rossman fold [38, 39].

In order to specify the role of AdhB in *B. cereus*, the virulence of the wild type and *ΔadhB* mutant was tested in an insect infection model. *G. mellonella* larvae were used as a model of infection as *B. cereus* is both a human and an insect pathogen [25, 44]. This study reveals that *adhB* plays an essential role during *B. cereus* virulence and could thus be considered as a new pathogenic factor.

During human or insect infections, *B. cereus* is able to resist the host immune system and persist. It can indeed survive phagocytosis by macrophages and can induce their apoptosis [20, 45]. The primary mechanism of macrophage-induced cytotoxicity is through the massive production of nitric oxide and oxidative stress at the peak of inflammation leading to bacterial death [46, 47]. Thus, bacterial response to NO is of major importance for bacterial survival and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO [48]. We have previously shown that *B. cereus* is particularly resistant to NO [15, 18, 45, 49]. Here, we show that the *ΔadhB* mutant was more sensitive than the wild-type strain to both oxidative and nitric stresses. Accordingly, this sensitivity may be implicated in the reduced mutant virulence in the insect model.

The initial step of bacterial response to NO and oxidative response is the detection of reactive oxygen and nitrogen species (ROS and RNS), which will permit to activate the detoxification and repair pathways. It has been previously shown that virulence factor production by *B. cereus* is dynamic and shaped by cellular oxidation [50]. ADH proteins have been previously shown to be involved in the reduction of alcohol and the production of NADH. NADPH is required to maintain and regenerate the cellular detoxifying and anti-oxidative defense systems [51]. The antioxidant defense system of *B. cereus* is constituted by an elaborate, often overlapping network of enzymes [52], but to the best of our knowledge, there was no evidence of ADH implication in the resistance of oxidative or NO stress. As oxidative and NO response overlap during the immune response, it is not surprising that mechanisms of bacterial resistance against ROS and RNS share similarities. The reduction capacity of ADH may be involved in NO detoxification. Bacterial capacity to detoxify NO through reduction is widely distributed in denitrifying bacteria but is also present in pathogens. For denitrifying bacteria, the reduction of nitrate to N₂ is part of the nitrogen cycle and prevents NO high toxicity; for pathogenic bacteria, NO detoxification might be a mean to survive under oxygen limited environments and to survive to nitrogen stress [46, 47, 53].

Taken together, we have identified a new virulence factor implicated in *B. cereus* resistance to host immunity whose activities may explain the pathogenic potential of clinical strains carrying this newly identified pathogenic biomarker.

Author Contributions

Conceptualization: Nalini Ramarao.

Formal analysis: Devon W. Kavanaugh, Nalini Ramarao.
**Funding acquisition:** Nalini Ramarao.

**Methodology:** Devon W. Kavanaugh, Constance Porrini, Rozenn Dervyn, Nalini Ramarao.

**Project administration:** Nalini Ramarao.

**Supervision:** Nalini Ramarao.

**Writing – original draft:** Devon W. Kavanaugh, Constance Porrini.

**Writing – review & editing:** Nalini Ramarao.

## References

1. Glasset B, Herbin S, Guiller L, Cadel-Six S, Vignaud ML, Grout J, et al. Large-scale survey of Bacillus cereus-induced food-borne outbreaks: epidemiologic and genetic characterization EuroSurveillance. 2016; 21(48): 30413.

2. Stenfors Arnesen L, Fagerlund A, Granum P. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev. 2008; 32:579-606. https://doi.org/10.1111/j.1574-6976.2008.00112.x PMID: 18422617

3. Ramarao N, Lereclus D, Sorokin A. The *Bacillus cereus* group. Molecular Medical Microbiology. 2015; III(Second Edition):1041-78.

4. Veysseyre F, Fourcade C, Lavigne JP, Sotto A. Bacillus cereus infection: 57 case patients and a literature review. Med Mal Infect. 2015; 45(11-12):436-40. Epub 2015/11/04. https://doi.org/10.1016/j.medmal.2015.09.011 PMID: 26525185

5. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 2010; 23(2):382-98. https://doi.org/10.1128/CMR.00073-09 PMID: 20375358

6. Ramarao N, Belotti L, Deboscker S, Ennahar-Vuillemin M, de Launay J, Lavigne T, et al. Two unrelated episodes of Bacillus cereus bacteremia in a neonatal intensive care unit. Am J Infect Control. 2014; 42 (6):694-5. https://doi.org/10.1016/j.ajic.2014.01.025 PMID: 24725514

7. Gaur AH, Patrick CC, McCullers JA, Flynn PM, Pearson TA, Razzouk BI, et al. Bacillus cereus bacteremia and meningitis in immunocompromised children. Clinic Infect dis. 2001; 32:1456-62. https://doi.org/10.1086/320154 PMID: 11317247

8. Lotte R, Herisse AL, Berrouane Y, Lotte L, Casagrande F, Landraud L, et al. Virulence Analysis of Bacillus cereus Isolated after Death of Preterm Neonates, Nice, France, 2013. Emerg Infect Dis. 2017; 23 (5):845-8. https://doi.org/10.3201/eid2305.161788 PMID: 28418291

9. Cormontagne D, Rigourd V, Vidic J, Rizzotto F, Bille E, Ramarao N. Bacillus cereus Induces Severe Infections in Preterm Neonates: Implication at the Hospital and Human Milk Bank Level. Toxins (Basel). 2021; 13(2). https://doi.org/10.3390/toxins13020123 PMID: 33562185

10. Hoffmaster AR, Hill KK, Gee JE, Marston CK, De BK, Popovic T, et al. Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J Clin Microbiol. 2006; 44(9):3352-60. 44/9/3352. https://doi.org/10.1128/JCM.00561-06 PMID: 16954272

11. Glasset B, Sperry M, Dervyn R, Herbin S, Brisabois A, Ramarao N. The cytotoxic potential of Bacillus cereus strains of various origins. Food Microbiol. 2021; 98:103759. https://doi.org/10.1016/j.fm.2021.103759 PMID: 33875199

12. Fagerlund A, Lindbäck T, Storset A, Granum P, Hardy S. Bacillus cereus Nhe is a pore forming toxin with structural and functional properties similar to ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia. Microbiol. 2008; 154:693-704. https://doi.org/10.1099/mic.0.2007/014134-0 PMID: 18310016

13. Ramarao N, Tran SL, Marin M, Vidic J. Advanced Methods for Detection of Bacillus cereus and Its Pathogenic Factors. Sensors (Basel). 2020; 20(9). https://doi.org/10.3390/s20092667 PMID: 32392794

14. Guinebretière MH, Thompson, Sorokin A, Normand P., Dawyndt P., Ehling-Schulz M, et al. Ecological diversification in the Bacillus cereus Group. Environ Microbiol. 2008; 10:851-65. https://doi.org/10.1111/j.1462-2920.2007.01495.x PMID: 18036180

15. Darrigo C, Guillemet E, Dervyn R, Ramarao N. The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway. PLoS ONE. 2016; 11(10):e0163321. https://doi.org/10.1371/journal.pone.0163321 PMID: 27711223
16. Guillemet E, Cadot C, Tran SL, Guinebretiere MH, Lereclus D, Ramarao N. The InhA metalloproteases of Bacillus cereus contribute concomitantly to virulence. J Bacteriol. 2010; 192(1):286-94. https://doi.org/10.1128/JB.00264-09 PMID: 19837797

17. Haydar A, Tran SL, Guillemet E, Darrigo C, Perchat S, Lereclus D, et al. InhA1-Mediated Cleavage of the Metalloprotease NprA Allows Bacillus cereus to Escape From Macrophages Front Microbiol. 2018; 23:1063. https://doi.org/10.3389/fmicb.2018.01063 PMID: 29875760

18. Guillemet E, Lereec A, Tran SL, Royer C, Barbosa I, Sansonetti P, et al. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response. Sci Rep. 2016; 6:29349. https://doi.org/10.1038/srep29349 PMID: 27435260

19. Tran SL, Guillemet E, Gohar M, Lereclus D, Ramarao N. CwpFM (EntFM) is a *Bacillus cereus* potential cell wall peptidase implicated in adhesion, biofilm formation and virulence. J Bacteriol. 2010; 192:2638-42. https://doi.org/10.1128/JB.01315-09 PMID: 20233921

20. Tran SL, Guillemet E, Ngo-Camus M, Clybouw C, Puhar A, Moris A, et al. Hemolysin II is a *Bacillus cereus* virulence factor that induces apoptosis of macrophages. Cell Microbiol. 2011; 13:92-108. https://doi.org/10.1111/j.1462-5822.2010.01522.x PMID: 20731668

21. Tran SL, Cormontagne D, Vidic J, Andre-Leroux G, Ramarao N. Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains. Toxins (Basel). 2020; 12(9). https://doi.org/10.3390/toxins12090593 PMID: 32937845

22. Glasset B, Herbin S, Granier S, Cavalié L, Lafeuille E, Guérin C, et al. Bacillus cereus, a serious cause of nosocomial infections: epidemiologic and genetic survey. PLoS ONE. 2018; 13(5):e0194346. https://doi.org/10.1371/journal.pone.0194346 PMID: 29791442

23. Guinebretière MH, Broussolle V, Nguyen-The C. Enterotoxigenic profiles of food-poisoning and food-borne *Bacillus cereus* strains. J Clin Microbiol. 2002; 40(8):3053-6. https://doi.org/10.1128/JCM.40.8.3053-3056.2002 PMID: 12149378

24. Martinez-Blanch JF, Sanchez G, Garay E, Aznar R. Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples. Int J Food Microbiol. 2009; 135(1):15-21. https://doi.org/10.1016/j.ijfoodmicro.2009.07.013 PMID: 19665814

25. Kamar R, Gohar M, Jéhanno I, Réjasse A, Kallassy M, Lereclus D, et al. Pathogenic Potential of Bacillus cereus Strains as Revealed by Phenotypic Analysis. J Clin Microbiol. 2013; 51:320-3. https://doi.org/10.1128/JCM.02848-12 PMID: 23135929

26. Kavanaugh D, Glasset B, Dervyn R, Guérin C, Plancade S, Cormontagne D, et al. New genetic biomarkers to differentiate pathogenic and clinically relevant Bacillus cereus strains. Clin Microb Infect. 2021. 7:S1198-743X(21)00283-4. https://doi.org/10.1016/j.cmi.2021.05.035 PMID: 34111580

27. Dervyn R, Kavanaugh DW, Cormontagne D, Glasset B, Ramarao N. Identification of a new pathogenicity island within the large pAH187_270 plasmid involved in *Bacillus cereus* virulence. Front. Cell. Infect. Microbiol. 2021; 11:788757.

28. Callejas-Negrete OA, Torres-Guzman JC, Padilla-Guerrero IE, Esquivel-Naranjo U, Padilla-Ballesteros MF, Garcia-Tapia A, et al. The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence. Microbiol Res. 2015; 172:57-67. https://doi.org/10.1016/j.micres.2014.11.006 PMID: 25534970

29. Saliola M, Falcone C. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation. Mol Gen Genet. 1995; 249(6):665-72. https://doi.org/10.1007/BF00418036 PMID: 8544832

30. Kelly JM, Drysdale MR, Sealy-Lewis HM, Jones IG, Lockington RA. Alcohol dehydrogenase III in Aspergillus nidulans is anaerobically induced and post-transcriptionally regulated. Mol Gen Genet. 1990; 222 (2-3):323-8. https://doi.org/10.1007/BF00633836 PMID: 2274033

31. Bakker BM, Bro C, Kotter P, Luttik MA, van Dijken JP, Pronk JT. The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae. J Bacteriol. 2000; 182 (17):4730-7. https://doi.org/10.1128/JB.182.17.4730-4737.2000 PMID: 10940011

32. Arnaud M, Chastanet A, Debarbouille M. New vector for efficient allelic replacement in naturally non-transformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol. 2004; 70(11):6887-91. https://doi.org/10.1128/AEM.70.11.6887-6891.2004 PMID: 15528558

33. Hagan CTt, Medik YB, Wang AZ. Nanotechnology Approaches to Improving Cancer Immunotherapy. Adv Cancer Res. 2018; 139:35-56. https://doi.org/10.1016/bs.acr.2018.05.003 PMID: 29941106

34. Ramarao N, Nielsen-LeRoux C, Lereclus D. The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis. J Vis Exp. 2012; 70:e4392. https://doi.org/10.3791/4392 PMID: 23271509
35. Tran S, Guillemet E, Lereclus D, Ramarao N. Iron regulates Bacillus thuringiensis haemolysin hlyII gene expression during insect infection. J Invert Pathol. 2013; 113:205–8. https://doi.org/10.1016/j.jip.2013.04.001 PMID: 23598183

36. Murzin AG. Structural classification of proteins: new superfamilies. Curr Opin Struct Biol. 1996; 6 (3):386–94. https://doi.org/10.1016/s0959-440x(96)80059-5 PMID: 8804825

37. Taneja B, Mande SC. Conserved structural features and sequence patterns in the GroES fold family. Protein Eng. 1999; 12(10):815–8. https://doi.org/10.1093/protein/12.10.815 PMID: 10556240

38. Rubach JK, Plapp BV. Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase. Biochemistry. 2003; 42(10):2907–15. https://doi.org/10.1021/bi0272656 PMID: 12627956

39. Thorn JM, Barton JD, Dixon NE, Ollis DL, Edwards KJ. Crystal structure of Escherichia coli QOR quinone oxidoreductase complexed with NADPH. J Mol Biol. 1995; 249(4):785–99. https://doi.org/10.1006/jmbi.1995.0337 PMID: 7602590

40. Mukherjee PK, Mohamed S, Chandra J, Kuhn D, Liu S, Antar OS, et al. Alcohol dehydrogenase restricts the ability of the pathogen Candida albicans to form a biofilm on catheter surfaces through an ethanol-based mechanism. Infect Immun. 2006; 74(7):3804–16. https://doi.org/10.1128/IAI.00161-06 PMID: 16790752

41. Plapp BV, Leidal KG, Murch BP, Green DW. Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats. Chem Biol Interact. 2015; 234:85–95. https://doi.org/10.1016/j.cbi.2014.12.040 PMID: 25641189

42. Jornvall H, Persson B, Jeffery J. Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem. 1987; 167(2):195–201. https://doi.org/10.1111/j.1432-1033.1987.tb13323.x PMID: 3622514

43. Sun HW, Plapp BV. Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J Mol Evol. 1992; 34(6):522–35. https://doi.org/10.1007/BF00160465 PMID: 1593644

44. Cadot C, Tran SL, Vignaud ML, De Buyser ML, Kolsto AB, Brisabois A, et al. InhA1, NprA and HlyII as candidates to differentiate pathogenic from non-pathogenic Bacillus cereus strains. J Clin Microbiol. 2010; 48:1358–65. https://doi.org/10.1128/JCM.02123-09 PMID: 20129969

45. Tran SL, Ramarao N. Bacillus cereus immune escape: a journey within macrophages. FEMS Microbiol Lett. 2013; 347:1–6. https://doi.org/10.1111/1574-6968.12209 PMID: 23827020

46. Porrini C, Ramarao N, Tran SL. Dr. NO and Mr. Toxic—the versatile role of nitric oxide. Biol Chem. 2020; 401(5):547–72. https://doi.org/10.1515/hsz-2019-0368 PMID: 31811798

47. Chin MP, Schauer DB, Deen WM. Nitric oxide, oxygen, and superoxide formation and consumption in macrophages and colonic epithelial cells. Chem Res Toxicol. 2010; 23(4):778–87. https://doi.org/10.1021/tx900415k PMID: 20201482

48. Zaki MH, Akuta T, Akaike T. Nitric oxide-induced nitrative stress involved in microbial pathogenesis. J Pharmacol Sci. 2005; 98(2):117–29. JST.JSTAGE/jphs/CRJ05004X. https://doi.org/10.1254/jphs.crj05004x PMID: 15937405

49. Porrini C, Guérin C, Tran SL, Dervyn R, Nicolas P, Ramarao N. Implication of a Key Region of Six *Bacillus cereus* Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress. Int J Mol Sci. 2021;11; 22(10):5079. https://doi.org/10.3390/ijms22105079 PMID: 34064887

50. Madeira JP, Alpha-Bazin B, Armengaud J, Duport C. Time dynamics of the Bacillus cereus exoproteome are shaped by cellular oxidation. Front Microbiol. 2015; 6:342. https://doi.org/10.3389/fmicb.2015.00342 PMID: 25954265

51. Agledal L, Niere M, Ziegler M. The phosphate makes a difference: cellular functions of NADP. Redox Rep. 2010; 15(1):2–10. https://doi.org/10.1179/174329210X12650506623122 PMID: 20196923

52. Duport C, Jobin M, Schmitt P. Adaptation in Bacillus cereus: From Stress to Disease. Front Microbiol. 2016; 7:1550. https://doi.org/10.3389/fmicb.2016.01550 PMID: 27757102

53. Leclerc M, Bedu-Ferrari C, Etienne-Mesmin L, Mariadassou M, Lebreuilly L, Tran SL, et al. Nitric Oxide Impacts Human Gut Microbiota Diversity and Functionalities. mSystems. 2021 Oct 26; 6(5):e0055821. https://doi.org/10.1128/mSystems.00558-21 PMID: 34519530