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.

Genetic diversity and population structure of Mycobacterium marinum: new insights into host and environmental specificities.

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.