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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., Author Summary Pathogenic bacteria have to resist host immune response and MgtC is used by several intracellular pathogens to promote bacterial multiplication inside macrophages. Here we investigated MgtC’s role in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in zebrafish embryos, but only in the presence of macrophages. Moreover, this mutant is more rapidly killed by macrophages than the wild-type strain. Both phenotypes can be mimicked upon production of a MgtC antagonistic peptide in wild-type Pseudomonas strain. MgtC thus provides a singular example of a virulence determinant that promotes strategies to subvert the antimicrobial behavior of macrophages, in both intracellular and extracellular pathogens and our results support an intramacrophage stage during in P. aeruginosa acute infection, as well as an interplay between MgtC role and phagosome acidification. In addition, P. aeruginosa MgtC is required for growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens, and limits biofilm formation. MgtC may share a similar function in intracellular and extracellular pathogens, with an outcome adapted to the different bacterial lifestyles

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.

Classically, pathogenic bacteria are classified as intracellular or extracellular pathogens. Intracellular bacterial pathogens, as Mycobacterium tuberculosis, Salmonella enterica, Brucella suis, or Listeria monocytogenes, can replicate within host cells. After entering the target host cell (professional or non-professional phagocyte), the intracellular pathogen follows vacuolar or cytosolic pathways to replicate. In contrast, extracellular pathogens, as Staphylococcus aureus, Pseudomonas aeruginosa or streptococci, avoid phagocytosis, thus promoting extracellular multiplication. However, the situation appears more complex with a dual lifestyle of intracellular/extracellular bacterial pathogens (Silva, 2012). Indeed, diverse bacterial intracellular pathogens have the ability to produce extracellular infections as a second phase after the initial intracellular stage. Conversely, several extracellular pathogens can enter host cells in vivo, resulting in a phase of intracellular residence, which can be of importance prior the typical extracellular infection. During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses that include oxidative and acid stresses, toxic metal cations, and antimicrobial peptides. Bacterial pathogens have developed strategies to escape the innate immune system and counteract the microbicidal action of macrophages (reviewed by Sarantis and Grinstein, 2012). Whereas some pathogens inhibit phagocytosis (extracellular pathogens), others use virulence factors to subvert the macrophage antimicrobial role and manipulate the host-cell to establish a replication niche. Hence, intracellular pathogens overcome macrophage defenses and use these immune cells as residence and dissemination strategies. Even if it accounts for a transitory initial event, so-called extracellular pathogens can also encounter an intramacrophage stage and have therefore to escape the killing by these immune cells. Intracellular bacteria use a broad range of molecular pathogenicity determinants to manipulate host cell processes and adapt to the intracellular environment. Recent data, described below, reveal that so-called extracellular pathogens have also acquired similar pathogenicity determinants to improve intramacrophage survival.

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.

Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans ( n = 68) and fish ( n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.