Contribution of intramacrophage stages to Pseudomonas aeruginosa infection outcome in zebrafish embryos: insights from mgtC and oprF mutants.

Pseudomonas aeruginosa often colonizes immunocompromised patients, causing acute and chronic infections. This bacterium can reside transiently inside cultured macrophages, but the contribution of the intramacrophic stage during infection remains unclear. MgtC and OprF have been identified as important bacterial factors when P. aeruginosa resides inside cultured macrophages. In this study, we showed that P. aeruginosa mgtC and oprF mutants, particular the latter one, had attenuated virulence in both mouse and zebrafish animal models of acute infection. To further investigate P. aeruginosa pathogenesis in zebrafish at a stage different from acute infection, we monitored bacterial load and visualized fluorescent bacteria in live larvae up to 4 days after infection. Whereas the attenuated phenotype of the oprF mutant was associated with a rapid elimination of bacteria, the mgtC mutant was able to persist at low level, a feature also observed with the wild-type strain in surviving larvae. Interestingly, these persistent bacteria can be visualized in macrophages of zebrafish. In a short-time infection model using a macrophage cell line, electron microscopy revealed that internalized P. aeruginosa wild-type bacteria were either released after macrophage lysis or remained intracellularly, where they were localized in vacuoles or in the cytoplasm. The mgtC mutant could also be detected inside macrophages, but without causing cell damage, whereas the oprF mutant was almost completely eliminated after phagocytosis, or localized in phagolysosomes. Taken together, our results show that the main role of OprF for intramacrophage survival impacts both acute and persistent infection by this bacterium. On the other hand, MgtC plays a clear role in acute infection but is not essential for bacterial persistence, in relation with the finding that the mgtC mutant is not completely eliminated by macrophages.

Structure-based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase.

Multidrug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to the failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+ -competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here, we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µm. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model.

Phylogenetic Groups and Antimicrobial Resistance Genes in Escherichia coli from Different Meat Species.

Escherichia coli isolated from meat of different animal species may harbour antimicrobial resistance genes and may thus be a threat to human health. The objectives of this study were to define antimicrobial resistance genes in E. coli isolates from pork, beef, chicken- and turkey meat and analyse whether their resistance genotypes associated with phylogenetic groups or meat species. A total number of 313 E. coli samples were isolated using standard cultural techniques. In 98% of resistant isolates, a dedicated resistance gene could be identified by PCR. Resistance genes detected were tet(A) and tet(B) for tetracycline resistance, strA and aadA1 for streptomycin resistance, sulI and sulII for resistance against sulphonamides, dfr and aphA for kanamycin resistance and blaTEM for ampicillin resistance. One stx1 harbouring E. coli isolated from pork harboured the tet(A) gene and belonged to phylogenetic group B2, whilst another stx1 positive isolate from beef was multi-resistant and tested positive for blaTEM,aphA, strA-B, sulII, and tet(A) and belonged to phylogenetic group A. In conclusion, the distribution of resistance elements was almost identical and statistically indifferent in isolates of different meat species. Phylogenetic groups did not associate with the distribution of resistance genes and a rather low number of diverse resistance genes were detected. Most E. coli populations with different resistance genes against one drug often revealed statistically significant different MIC values.

Zebrafish Embryo Infection Model to Investigate Pseudomonas aeruginosa Interaction With Innate Immunity and Validate New Therapeutics.

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected patients with cystic fibrosis (CF). Considering the intrinsic and acquired resistance of P. aeruginosa to currently used antibiotics, new therapeutic strategies against this pathogen are urgently needed. Whereas virulence factors of P. aeruginosa are well characterized, the interplay between P. aeruginosa and the innate immune response during infection remains unclear. Zebrafish embryo is now firmly established as a potent vertebrate model for the study of infectious human diseases, due to strong similarities of its innate immune system with that of humans and the unprecedented possibilities of non-invasive real-time imaging. This model has been successfully developed to investigate the contribution of bacterial and host factors involved in P. aeruginosa pathogenesis, as well as rapidly assess the efficacy of anti-Pseudomonas molecules. Importantly, zebrafish embryo appears as the state-of-the-art model to address in vivo the contribution of innate immunity in the outcome of P. aeruginosa infection. Of interest, is the finding that the zebrafish encodes a CFTR channel closely related to human CFTR, which allowed to develop a model to address P. aeruginosa pathogenesis, innate immune response, and treatment evaluation in a CF context.

A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo.

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

Pseudomonas aeruginosa OprF plays a role in resistance to macrophage clearance during acute infection.

While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models. However, the role of macrophages in P. aeruginosa clearance in vivo remains poorly studied. The major outer membrane porin OprF has been recently shown to be involved in P. aeruginosa fate within cultured macrophages and analysis of an oprF mutant may thus provide insights to better understand the relevance of this intramacrophage stage during infection. In the present study, we investigated for the first time the virulence of a P. aeruginosa oprF mutant in a vertebrate model that harbors functional macrophages, the zebrafish (Danio rerio) embryo, which offers powerful tools to address macrophage-pathogen interactions. We established that P. aeruginosa oprF mutant is attenuated in zebrafish embryos in a macrophage-dependent manner. Visualization and quantification of P. aeruginosa bacteria phagocytosed by macrophages after injection into closed cavities suggested that the attenuated phenotype of oprF mutant is not linked to higher macrophage recruitment nor better phagocytosis than wild-type strain. Using cultured macrophages, we showed an intramacrophage survival defect of P. aeruginosa oprF mutant, which is correlated with elevated association of bacteria with acidic compartments. Notably, treatment of embryos with bafilomycin, an inhibitor of acidification, increased the sensibility of embryos towards both wild-type and oprF mutant, and partially suppressed the attenuation of oprF mutant. Taken together, this work supports zebrafish embryo as state-of-the-art model to address in vivo the relevance of P. aeruginosa intramacrophage stage. Our results highlight the contribution of macrophages in the clearance of P. aeruginosa during acute infection and suggest that OprF protects P. aeruginosa against macrophage clearance by avoiding bacterial elimination in acidified phagosomes.

How Pathogens Feel and Overcome Magnesium Limitation When in Host Tissues.

Host organisms utilize nutritional immunity to limit the availability of nutrients essential to an invading pathogen. Nutrients may include amino acids, nucleotide bases, and transition metals, the essentiality of which varies among pathogens. The mammalian macrophage protein Slc11a1 (previously Nramp1) mediates resistance to several intracellular pathogens. Slc11a1 is proposed to restrict growth of Salmonella enterica serovar Typhimurium in host tissues by causing magnesium deprivation. This is intriguing because magnesium is the most abundant divalent cation in all living cells. A pathogen's response to factors such as Slc11a1 that promote nutritional immunity may therefore reflect what the pathogen 'feels' in its cytoplasm, rather than the nutrient concentration in host cell compartments.

Synthetic hydrophobic peptides derived from MgtR weaken Salmonella pathogenicity and work with a different mode of action than endogenously produced peptides.

Due to the antibiotic resistance crisis, novel therapeutic strategies need to be developed against bacterial pathogens. Hydrophobic bacterial peptides (small proteins under 50 amino acids) have emerged as regulatory molecules that can interact with bacterial membrane proteins to modulate their activity and/or stability. Among them, the Salmonella MgtR peptide promotes the degradation of MgtC, a virulence factor involved in Salmonella intramacrophage replication, thus providing the basis for an antivirulence strategy. We demonstrate here that endogenous overproduction of MgtR reduced Salmonella replication inside macrophages and lowered MgtC protein level, whereas a peptide variant of MgtR (MgtR-S17I), which does not interact with MgtC, had no effect. We then used synthetic peptides to evaluate their action upon exogenous addition. Unexpectedly, upon addition of synthetic peptides, both MgtR and its variant MgtR-S17I reduced Salmonella intramacrophage replication and lowered MgtC and MgtB protein levels, suggesting a different mechanism of action of exogenously added peptides versus endogenously produced peptides. The synthetic peptides did not act by reducing bacterial viability. We next tested their effect on various recombinant proteins produced in Escherichia coli and showed that the level of several inner membrane proteins was strongly reduced upon addition of both peptides, whereas cytoplasmic or outer membrane proteins remained unaffected. Moreover, the α-helical structure of synthetic MgtR is important for its biological activity, whereas helix-helix interacting motif is dispensable. Cumulatively, these results provide perspectives for new antivirulence strategies with the use of peptides that act by reducing the level of inner membrane proteins, including virulence factors.

Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants.

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.

Activity of a Synthetic Peptide Targeting MgtC on Pseudomonas aeruginosa Intramacrophage Survival and Biofilm Formation.

Antivirulence strategies aim to target pathogenicity factors while bypassing the pressure on the bacterium to develop resistance. The MgtC membrane protein has been proposed as an attractive target that is involved in the ability of several major bacterial pathogens, including Pseudomonas aeruginosa, to survive inside macrophages. In liquid culture, P. aeruginosa MgtC acts negatively on biofilm formation. However, a putative link between these two functions of MgtC in P. aeruginosa has not been experimentally addressed. In the present study, we first investigated the contribution of exopolysaccharides (EPS) in the intramacrophage survival defect and biofilm increase of mgtC mutant. Within infected macrophages, expression of EPS genes psl and alg was increased in a P. aeruginosa mgtC mutant strain comparatively to wild-type strain. However, the intramacrophage survival defect of mgtC mutant was not rescued upon introduction of psl or alg mutation, suggesting that MgtC intramacrophage role is unrelated to EPS production, whereas the increased biofilm formation of mgtC mutant was partially suppressed by introduction of psl mutation. We aimed to develop an antivirulence strategy targeting MgtC, by taking advantage of a natural antagonistic peptide, MgtR. Heterologous expression of mgtR in P. aeruginosa PAO1 was shown to reduce its ability to survive within macrophages. We investigated for the first time the biological effect of a synthetic MgtR peptide on P. aeruginosa. Exogenously added synthetic MgtR peptide lowered the intramacrophage survival of wild-type P. aeruginosa PAO1, thus mimicking the phenotype of an mgtC mutant as well as the effect of endogenously produced MgtR peptide. In correlation with this finding, addition of MgtR peptide to bacterial culture strongly reduced MgtC protein level, without reducing bacterial growth or viability, thus differing from classical antimicrobial peptides. On the other hand, the addition of exogenous MgtR peptide did not affect significantly biofilm formation, indicating an action toward EPS-independent phenotype rather than EPS-related phenotype. Cumulatively, our results show an antivirulence action of synthetic MgtR peptide, which may be more potent against acute infection, and provide a proof of concept for further exploitation of anti-Pseudomonas strategies.

PtpA, a secreted tyrosine phosphatase from Staphylococcus aureus, contributes to virulence and interacts with coronin-1A during infection.

Secretion of bacterial signaling proteins and adaptation to the host, especially during infection, are processes that are often linked in pathogenic bacteria. The human pathogen Staphylococcus aureus is equipped with a large arsenal of immune-modulating factors, allowing it to either subvert the host immune response or to create permissive niches for its survival. Recently, we showed that one of the low-molecular-weight protein tyrosine phosphatases produced by S. aureus, PtpA, is secreted during growth. Here, we report that deletion of ptpA in S. aureus affects intramacrophage survival and infectivity. We also observed that PtpA is secreted during macrophage infection. Immunoprecipitation assays identified several host proteins as putative intracellular binding partners for PtpA, including coronin-1A, a cytoskeleton-associated protein that is implicated in a variety of cellular processes. Of note, we demonstrated that coronin-1A is phosphorylated on tyrosine residues upon S. aureus infection and that its phosphorylation profile is linked to PtpA expression. Our results confirm that PtpA has a critical role during infection as a bacterial effector protein that counteracts host defenses.

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication.

Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties.

MgtC as a Host-Induced Factor and Vaccine Candidate against Mycobacterium abscessus Infection.

Mycobacterium abscessus is an emerging pathogenic mycobacterium involved in pulmonary and mucocutaneous infections, presenting a serious threat for patients with cystic fibrosis (CF). The lack of an efficient treatment regimen and the emergence of multidrug resistance in clinical isolates require the development of new therapeutic strategies against this pathogen. Reverse genetics has revealed genes that are present in M. abscessus but absent from saprophytic mycobacteria and that are potentially involved in pathogenicity. Among them, MAB_3593 encodes MgtC, a known virulence factor involved in intramacrophage survival and adaptation to Mg(2+) deprivation in several major bacterial pathogens. Here, we demonstrated a strong induction of M. abscessus MgtC at both the transcriptional and translational levels when bacteria reside inside macrophages or upon Mg(2+) deprivation. Moreover, we showed that M. abscessus MgtC was recognized by sera from M. abscessus-infected CF patients. The intramacrophage growth (J774 or THP1 cells) of a M. abscessus knockout mgtC mutant was, however, not significantly impeded. Importantly, our results indicated that inhibition of MgtC in vivo through immunization with M. abscessus mgtC DNA, formulated with a tetrafunctional amphiphilic block copolymer, exerted a protective effect against an aerosolized M. abscessus challenge in CF (ΔF508 FVB) mice. The formulated DNA immunization was likely associated with the production of specific MgtC antibodies, which may stimulate a protective effect by counteracting MgtC activity during M. abscessus infection. These results emphasize the importance of M. abscessus MgtC in vivo and provide a basis for the development of novel therapeutic tools against pulmonary M. abscessus infections in CF patients.

Use of the Salmonella MgtR peptide as an antagonist of the Mycobacterium MgtC virulence factor.

BACKGROUND: The MgtC virulence factor has been proposed as an attractive target for antivirulence strategies because it is shared by several important bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis (Mtb). AIM: A natural antagonistic peptide, MgtR, which interacts with MgtC and modulates its stability, has been identified in Salmonella, and we investigated its efficiency to target MgtC in another pathogen. MATERIALS & METHODS: We evaluated the interaction between Salmonella MgtR peptide and the Mtb MgtC protein using an in vivo bacterial two-hybrid system and we addressed the effect of exogenously added synthetic MgtR and endogenously expressed peptide. RESULTS: MgtR peptide strongly interacted with Mtb MgtC protein and exogenously added synthetic MgtR peptide-reduced Mtb MgtC level and interfered with the dimerization of Mtb MgtC. Importantly, heterologous expression of MgtR in Mycobacterium bovis BCG resulted in increased phagocytosis and reduced intramacrophage survival. CONCLUSION: MgtR peptide can target Mtb MgtC protein and reduce mycobacterial macrophage resistance, thus providing a promising new scaffold for the development of antivirulence compounds.

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.

Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way.

Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosamgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

Mycobacterium marinum MgtC plays a role in phagocytosis but is dispensable for intracellular multiplication.

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg2+ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg2+ deprivation and is required for optimal growth in Mg2+-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg2+ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen's lifestyles.

Overexpression of the Salmonella KdpF membrane peptide modulates expression of kdp genes and intramacrophage growth.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, including transporters and sensor kinases. The KdpF peptide, which is cotranscribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s). The mycobacterial KdpF can interact with the KdpD histidine kinase, and kdpF overexpression has been shown to reduce intramacrophage replication of Mycobacterium bovis BCG. In this study, we investigated whether KdpF displays similar behavior in another intracellular pathogen, Salmonella enterica serovar Typhimurium. We show that Salmonella KdpF can interact with KdpD in a bacterial two-hybrid assay. We have constructed a Salmonella strain overexpressing kdpF, and we have investigated expression of the kdp regulon, as well as intramacrophage survival. We show that kdpF overexpression reduces expression of kdpA and kdpD genes under potassium limitation. Moreover, kdpF overexpression increases intramacrophage multiplication of S. Typhimurium. Hence, our results indicate that KdpF can play a regulatory role in S. Typhimurium, modulating kdp gene expression and intramacrophage survival, but in a way that differs from the one reported for M. bovis BCG.

Overexpression of the KdpF membrane peptide in Mycobacterium bovis BCG results in reduced intramacrophage growth and altered cording morphology.

Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, such as sensor kinases. To date, regulatory membrane peptides have been completely overlooked in mycobacteria. The 30 amino-acid-long KdpF peptide, which is co-transcribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s) towards the KdpD sensor kinase. Herein, we showed by quantitative RT-PCR that the Mycobacterium bovis BCG kdpAB and kdpDE genes clusters are differentially induced in potassium-deprived broth medium or within infected macrophages. We have overexpressed the kdpF gene in M. bovis BCG to investigate its possible regulatory role and effect on mycobacterial virulence. Our results indicate that KdpF does not play a critical regulatory role on kdp genes expression despite the fact that KdpF interacts with the KdpD sensor kinase in a bacterial two-hybrid assay. However, overexpression of kdpF results in a significant reduction of M. bovis BCG growth in both murine and human primary macrophages, and is associated with a strong alteration of colonial morphology and impaired cording formation. To identify novel KdpF interactants, a mycobacterial library was screened using KdpF as bait in the bacterial two-hybrid system. This allowed us to identify members of the MmpL family of membrane proteins, known to participate in the biosynthesis/transport of various cell wall lipids, thus highlighting a possible link between KdpF and cell wall lipid metabolism. Taken together, these data suggest that KdpF overexpression reduces intramacrophage growth which may result from alteration of the mycobacterial cell wall.