The bacterial Mfd protein prevents DNA damage induced by the host nitrogen immune response in a NER-independent but RecBC-dependent pathway

Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway

In the context of NO stress, we show that <i>mfd</i> and <i>recBC</i>/<i>addAB</i> deletions are epistatic.

<p>As RecBC participates in the repair of Double Strand Breaks (DSB), this indicates that Mfd also participates in DSB repair. It is unlikely that Mfd prevents DSBs because, in that case, RecBC would be essential for DNA repair in a <i>mfd</i> mutant. By contrast, we hypothesized that following the DSBs induced by NO exposure, RecBC has not directly access to Double strand ends (DSEs). Thus, Mfd would act first by removing the RNAP blocked on DNA lesions. Then the RecBCD complex can be recruited to repairs the DSB. RecBCD unwinds the DNA helix and degrades single strands. When RecBDC encounters a Chi site on the DNA, the degradation of the 5’ terminus is enhanced leaving a 3’ overhang and leads to the formation of an ssDNA. RecA binds to ssDNA and promotes repair by recombination with a homologous molecule of DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163321#pone.0163321.ref050" target="_blank">50</a>].</p

Mfd confers resistance to NO stress.

<p>(A) Mice peritoneal macrophages isolated from wild type (BM wt) and NOS2-/- (BM KO) mice were infected with <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains. At the indicated time points, bacterial survival was calculated by plating and normalized to the cfu obtained at t0. Results represent mean values of at least 3 independent experiments done in triplicates. (B) Bacteria were exposed directly to chemically generated NO (1.5 mM sodium nitrite) in a cell free system for the indicated times. The bacteria were then harvested and plated on agar plates to evaluate bacterial survival. Results represent mean values of at least 3 independent experiments done in triplicates.</p

Phenotypical analysis of the Δ<i>mfd</i> mutant in mutagenic conditions.

<p>(A) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were inoculated in LB medium at a starting optical density (OD) of 0.07 and grown at 25°C with agitation. The OD was measured every hour at 600 nm. This graph represents representative growth curves out of at least five independent experiments. (B) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates containing 50 ng/mL mitomycin C. Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (C) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates and exposed to UV light for 0 to 15 seconds at 5J/m². Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (D) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown in LB medium until entry into stationary growth phase. Culture supernatant was filtered and added to HeLa cells. Cytotoxicity was measured by the trypan blue method after 2 h of incubation. Results are means of three independent experiments. (E) Bacterial strains were grown in LB medium at 37°C under agitation or without agitation (semi anaerobiosis). This graph represents representative growth curves out of at least three independent experiments. (F) Bacterial strains were grown in LB medium at 37°C under agitation, then diluted with the pH adjusted to 5, 6 or 7. Cfu were calculated after 24 h of growth by plating serial dilutions on LB agar plates. Results are means of at least three independent experiments. (G) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were cultured in LB medium for 24 h in the presence of the anti microbial peptide cecropin A. Cfu were calculated by plating serial dilutions on LB agar plates. Results are means of three independent experiments.</p

NO IC 50 on <i>B</i>. <i>cereus</i> mutants.

<p>NO IC 50 on <i>B</i>. <i>cereus</i> mutants.</p

Role of Mfd in <i>in vivo</i> survival following NO stress.

<p>C57/Bl6/Sev 129 mice (Wt Mice) and NOS2-/- mice (iNOS-KO mice) were inoculated intranasally with <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant bacteria (5.10⁶/mice). Wild type and <i>Δmfd</i> mutant strains were recovered from the lung (A) and from the brain (B) following mice death. The cfu recovered post infection was calculated by plating the bacteria on LB agar plates. For each mouse, the same symbol is used for lung (A) and brain (B) values. Cfu is shown for 5 wild type mice infected with Bc407, 4 wild type mice infected with Δ<i>mfd</i>, 3 KO mice infected with Bc407 and 3 KO mice infected with Δ<i>mfd</i>.</p

The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway - Fig 4

<p><b>(A) AddAB but not UvrA is involved to prevent NO stress</b>. <i>B</i>. <i>cereus</i> wild type and mutant strains were exposed to 1.5 mM NO for 1 h in a cell-free system. Bacteria were harvested and plated on agar plates to evaluate bacterial survival. Cfu counts were normalized to initial cfu. The results reported are mean values of at least three independent experiments. P values are calculated using the Student test. <b>(B) Survival of <i>B</i>. <i>cereus</i> mutant strains with Raw cells.</b> Raw cells were infected with <i>B</i>. <i>cereus</i> wild type and mutant strains at a multiplicity of infection of 10 at 37°C. After 20 h of incubation, bacteria were recovered by scraping and counted by plating serial dilutions on agar plates. Alternatively, the NO inhibitor NMMLA was added to the cells at 1 mM.</p

Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices

InhA1-mediated cleavage of the metalloprotease NprA allows Bacillus cereus to escape from macrophages

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation in the host despite the induction of inflammation. To circumvent inflammation, bacteria must resist the bactericidal activity of professional phagocytes, which constitute a first line of host defense against pathogens. Interactions between phagocytic cells and B. cereus are still poorly characterized and the mechanism of resistance to the host immune system is not known yet. We have previously shown that the spores are phagocytosed by macrophages but survive and escape from these cells. The metalloprotease InhA1 is a key effector involved in these processes. inhA1-deficient spores are retained intracellularly, in contrast to the wild type strain spores. NprA is also a B. cereus metalloprotease able to cleave tissue components such as fibronectin, laminin, and collagen. Here, we show that NprA, concomitantly secreted with InhA1 in the B. cereus secretome, is essential to promote bacterial escape from macrophages. We show that InhA1 cleaves NprA at specific sites. This cleavage allows liberation of the mature form of the NprA protein in the supernatant of the wild type strain. This mature form of NprA is actually the principal effector allowing bacterial escape from host macrophages