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IntroductionThis study identifies Staphylococcus debuckii as a new coagulase-negative staphylococcal species isolated from diabetic foot osteomyelitis (DFOM) and provides an in-depth analysis of its pathogenic and virulence profile, as well as demonstrating its potential to cause infection.MethodsThe S. debuckii NSD001 strain was examined for its planktonic growth, biofilm production, and phagocytosis rates in murine macrophages compared to S. aureus NSA739. Additionally, persistence and replication within human osteoblasts were investigated, while the zebrafish embryo model was employed to assess virulence. Genomic sequencing and bioinformatic analysis were also conducted to identify genes associated with virulent potential.Results and DiscussionS. debuckii NSD001 exhibited robust planktonic growth and significant biofilm production, highlighting its capacity to initiate and maintain an infection, and demonstrated similar rates of phagocytosis as S. aureus NSA739 in murine macrophages, suggesting a mechanism for evading initial host defenses. The strain persisted and replicated within human osteoblasts, indicative of a strategy for intracellular survival and facilitation of chronic osteomyelitis. The zebrafish embryo model revealed a slower, yet fatal, virulence profile for S. debuckii NSD001 compared to the rapid lethality induced by S. aureus NSA739. Genomic sequencing and bioinformatic analysis uncovered various genes corroborating its virulence. S. debuckii NSD001 poses a significant concern in DFOM due to its ability to form biofilms and survive within host cells, presenting challenges for current treatment strategies. This underscores the need for updated clinical protocols and increased awareness among healthcare professionals to effectively manage infections caused by this emerging pathogen.

ABSTRACT Staphylococcus aureus is a human pathogen that is extremely adaptable and is the cause of a variety of nosocomial and community-acquired infectious diseases. During infection, S. aureus is able to affect the host cell in many ways to enable its own multiplication, spread, and evasion of the host immune defense. One of the mechanisms utilized by S. aureus to survive is to inhibit the SUMOylation of host proteins in order to increase its intracellular survival and persistence. Here, we show that the reduction in the levels of cellular SUMO-conjugated proteins in S. aureus strain Newman-infected RAW 264.7 cells is associated with the S. aureus secreted protein tyrosine phosphatase PtpA, which results in a reduction of ubiquitin-conjugating enzyme 9 (Ubc9) protein level, the critical enzyme of the SUMOylation modification. In addition, we demonstrate that the amino acid residue D120, which is essential for PtpA phosphatase activity, is required for this reduction. This study shows for the first time that S. aureus strain Newman impedes via PtpA the host SUMOylation response, which contributes to promoting the persistence of S. aureus within the host. IMPORTANCE Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

Staphylococcus aureus (S. aureus) is a common and virulent human pathogen causing several serious illnesses including skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome. Antibiotics were first introduced in the 1940s, leading to the belief that bacterial illnesses would be eradicated. However, microorganisms, including S. aureus, began to develop antibiotic resistance from the increased use and abuse of antibiotics. Antibiotic resistance is now one of the most serious threats to global public health. Bacteria like methicillin-resistant Staphylococcus aureus (MRSA) remain a major problem despite several efforts to find new antibiotics. New treatment approaches are required, with bacteriophage treatment, a non-antibiotic strategy to treat bacterial infections, showing particular promise. The ability of S. aureus to resist a wide range of antibiotics makes it an ideal candidate for phage therapy studies. Bacteriophages have a relatively restricted range of action, enabling them to target pathogenic bacteria. Their usage, usually in the form of a cocktail of bacteriophages, allows for more focused treatment while also overcoming the emergence of resistance. However, many obstacles remain, particularly in terms of their effects in vivo, necessitating the development of animal models to assess the bacteriophage efficiency. Here, we provide a review of the animal models, the various clinical case treatments, and clinical trials for S. aureus phage therapy.

Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium. The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies.

Staphylococcus aureus is one of the main pathogens isolated from diabetic foot infections (DFI). The purpose of this study was to evaluate the importance of the persistence of S. aureus in this environment and the possible modifications of the bacterial genome content over time. Molecular typing of S. aureus isolates cultured from patients with the same DFI over a 7-year study revealed a 25% rate of persistence of this species in 48 patients, with a short median persistence time of 12weeks (range: 4–52weeks). Non-specific clonal complexes were linked to this persistence. During the follow-up, bla genes were acquired in three cases, whereas some virulence markers were lost in all cases after a long period of colonization (21.5weeks). Only one patient (2%) had a long-term persistence of 48weeks. The genome sequencing of a clonal pair of early/late strains isolated in this patient showed mutations in genes encoding bacterial defence and two-component signal transduction systems. Although, this study suggests that the long-term persistence of S. aureus in DFI is a rare event, genomic evolution is observed, highlighting the low adaptive ability of S. aureus to the specific environment and stressful conditions of diabetic foot ulcers. These results provide the basis for better understanding of S. aureus dynamics during persistent colonization in chronic wounds.

Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

Staphylococcus aureus is a clinically important pathogen that causes a wide range of human infections, from minor skin infections to severe tissue infection and sepsis. S. aureus has a high level of antibiotic resistance and is a common cause of infections in hospitals and the community. The rising prevalence of community-acquired methicillin-resistant S. aureus (CA-MRSA), combined with the important severity of S. aureus infections in general, has resulted in the frequent use of anti-staphylococcal antibiotics, leading to increasing resistance rates. Antibiotic-resistant S. aureus continues to be a major health concern, necessitating the development of novel therapeutic strategies. S. aureus uses a wide range of virulence factors, such as toxins, to develop an infection in the host. Recently, anti-virulence treatments that directly or indirectly neutralize S. aureus toxins have showed promise. In this review, we provide an update on toxin pathogenic characteristics, as well as anti-toxin therapeutical strategies.

Abstract Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in Vmax for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

Anthrax bacteria: a step in the pathway to biofilms An enzyme that adds phosphate groups to other proteins, PrkC, mediates molecular signaling events that allow anthrax bacteria to form biofilms. Bacillus anthracis is widely used as a model to explore the formation of biofilms that allows many bacterial infections to resist immune defenses. An international research team led by Yogendra Singh and Andaleeb Sajid at the CSIR-Institute of Genomics and Integrative Biology in Delhi, India, studied the bacterial protein kinase PrkC. The researchers found that PrkC phosphorylates a “chaperone” protein that assist the assembly and disassembly of other protein-based structures. This signaling protein and the chaperone help in biofilm formation. Establishing this link in the signaling chain leading to biofilms will guide future research to combat the role of biofilms in disease.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of “neutrophil extracellular traps” (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways.

ABSTRACT How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division. IMPORTANCE Bacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.