Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate.

According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis.

Phosphorus (P) is an essential component of core biological molecules. In bacteria, P is acquired mainly as inorganic orthophosphate (Pi) and assimilated into adenosine triphosphate (ATP) in the cytoplasm. Although P is essential, excess cytosolic Pi hinders growth. We now report that bacteria limit Pi uptake to avoid disruption of Mg2+-dependent processes that result, in part, from Mg2+ chelation by ATP. We establish that the MgtC protein inhibits uptake of the ATP precursor Pi when Salmonella enterica serovar Typhimurium experiences cytoplasmic Mg2+ starvation. This response prevents ATP accumulation and overproduction of ribosomal RNA that together ultimately hinder bacterial growth and result in loss of viability. Even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake increases ATP synthesis, depletes free cytoplasmic Mg2+, inhibits protein synthesis, and hinders growth. Our results provide a framework to understand the molecular basis for Pi toxicity. Furthermore, they suggest a regulatory logic that governs P assimilation based on its intimate connection to cytoplasmic Mg2+ homeostasis.

A protein that controls the onset of a Salmonella virulence program.

The mechanism of action and contribution to pathogenesis of many virulence genes are understood. By contrast, little is known about anti-virulence genes, which contribute to the start, progression, and outcome of an infection. We now report how an anti-virulence factor in Salmonella enterica serovar Typhimurium dictates the onset of a genetic program that governs metabolic adaptations and pathogen survival in host tissues. Specifically, we establish that the anti-virulence protein CigR directly restrains the virulence protein MgtC, thereby hindering intramacrophage survival, inhibition of ATP synthesis, stabilization of cytoplasmic pH, and gene transcription by the master virulence regulator PhoP. We determine that, like MgtC, CigR localizes to the bacterial inner membrane and that its C-terminal domain is critical for inhibition of MgtC. As in many toxin/anti-toxin genes implicated in antibiotic tolerance, the mgtC and cigR genes are part of the same mRNA. However, cigR is also transcribed from a constitutive promoter, thereby creating a threshold of CigR protein that the inducible MgtC protein must overcome to initiate a virulence program critical for pathogen persistence in host tissues.

A trans-acting leader RNA from a Salmonella virulence gene.

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

The malS-5'UTR weakens the ability of Salmonella enterica serovar Typhi to survive in macrophages by increasing intracellular ATP levels.

Bacterial non-coding RNAs (ncRNAs), as important regulatory factors, are involved in many cellular processes, including virulence and protection against environmental stress. The 5' untranslated region (UTR) of malS (named malS-5'UTR), a regulatory ncRNA, increases the invasive capacity and influences histidine biosynthesis in Salmonella enterica serovar Typhi (S. Typhi). In this study, we found that overexpression of the malS-5'UTR decreased S. Typhi survival within macrophages. A microarray analysis of a strain overexpressing the malS-5'UTR revealed a significant increase in the mRNA levels of the atp operon. The intracellular ATP levels were elevated in the malS-5'UTR overexpression strain. Quantitative real-time polymerase chain reaction results showed that the malS-5'UTR downregulated the mRNA levels of phoP, phoQ, and mgtC. MgtC, its expression is regulated by PhoP/PhoQ two-component regulatory system, inhibits the F1F0 ATP synthase, thereby preventing the accumulation of ATP to non-physiological levels and the acidification of the cytoplasm within macrophages. Thus, we propose that the malS-5'UTR weakens the ability of S. Typhi to survive in macrophages, probably because of the accumulation of ATP within macrophages, by regulating the mRNA levels of mgtC and the atp operon in a phoP-dependent manner.

Flagella-independent surface motility in Salmonella enterica serovar Typhimurium.

Flagella are multiprotein complexes necessary for swimming and swarming motility. In Salmonella enterica serovar Typhimurium, flagella-mediated motility is repressed by the PhoP/PhoQ regulatory system. We now report that Salmonella can move on 0.3% agarose media in a flagella-independent manner when experiencing the PhoP/PhoQ-inducing signal low Mg(2+). This motility requires the PhoP-activated mgtA, mgtC, and pagM genes, which specify a Mg(2+) transporter, an inhibitor of Salmonella's own F1Fo ATPase, and a small protein of unknown function, respectively. The MgtA and MgtC proteins are necessary for pagM expression because pagM mRNA levels were lower in mgtA and mgtC mutants than in wild-type Salmonella, and also because pagM expression from a heterologous promoter rescued motility in mgtA and mgtC mutants. PagM promotes group motility by a surface protein(s), as a pagM-expressing strain conferred motility upon a pagM null mutant, and proteinase K treatment eliminated motility. The pagM gene is rarely found outside subspecies I of S. enterica and often present in nonfunctional allelic forms in organisms lacking the identified motility. Deletion of the pagM gene reduced bacterial replication on 0.3% agarose low Mg(2+) media but not in low Mg(2+) liquid media. Our findings define a form of motility that allows Salmonella to scavenge nutrients and to escape toxic compounds in low Mg(2+) semisolid environments.

An RNA motif advances transcription by preventing Rho-dependent termination.

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.

Enhanced succinic acid productivity by expression of mgtCB gene in Escherichia coli mutant.

In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg(2+) concentration and mgtB to enhance the transport of Mg(2+) into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)2 and NH3·H2O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L(-1) h(-1)) compared with that by using the engineering strain with the overexpression of mgtA gene.

Impact of MgtC on the Fitness of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an extracellular foodborne pathogen and usually causes self-limiting diarrhea in healthy humans. MgtC is known as a key subversion factor that contributes to intramacrophage adaptation and intracellular survival in certain important pathogens. Whether MgtC influences the fitness of Y. pseudotuberculosis is unclear. According to in silico analysis, MgtC in Y. pseudotuberculosis might share similar functions with other bacterial pathogens, such as Salmonella. Studies indicated that MgtC was clearly required for Y. pseudotuberculosis growth in vitro and bacterial survival in macrophages under Mg2+ starvation. Transcriptome analysis by RNA-seq indicated that 127 differentially expressed genes (DEGs) (fold change > 2 and p < 0.001) were discovered between wild-type PB1+ and mgtC mutant inside macrophages. However, a lack of MgtC only moderately, albeit significantly, reduced the virulence of Y. pseudotuberculosis in mice. Overall, this study provides additional insights for the role of MgtC in Y. pseudotuberculosis.

The mgtCBR mRNA Leader Secures Growth of Salmonella in Both Host and Non-host Environments.

Upon intracellular cues, bacterial mRNA leaders often form secondary structures that determine expression of a downstream protein-coding region(s), thereby providing bacteria with a mechanism to control the amounts of necessary proteins in the right locales. Here we describe a polycistronic mRNA leader that secures bacterial growth by preventing dysregulated expression of the protein-coding regions. In Salmonella, the mgtCBR mRNA encodes the virulence protein MgtC and the Mg2+ transporter MgtB. A mutant designed to produce leaderless mgtCBR mRNA induced MgtC and MgtB in conditions that promote mgtC transcription. The dysregulated expression of MgtC and MgtB impaired bacterial growth under all such non-host environments. While MgtC, but not MgtB, normally reduces ATP levels in a process requiring the F1F0 ATP synthase, dysregulated MgtC and MgtB reduced ATP levels independently of the F1F0 ATP synthase, which correlated with the mutant's growth defect. The mutant showed dysregulated MgtC expression and attenuated survival inside macrophages. While MgtB normally does not affect the phenotype, MgtB impaired intramacrophage survival of the mutant in the presence of MgtC. We provide an example showing that a polycistronic mRNA leader prevents the dysregulated function of protein-coding regions to allow bacteria to proliferate across complex niches.

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.

A rule governing the FtsH-mediated proteolysis of the MgtC virulence protein from Salmonella enterica serovar Typhimurium.

A tightly controlled turnover of membrane proteins is required for lipid bilayer stability, cell metabolism, and cell viability. Among the energy-dependent AAA+ proteases in Salmonella, FtsH is the only membrane-bound protease that contributes to the quality control of membrane proteins. FtsH preferentially degrades the C-terminus or N-terminus of misfolded, misassembled, or damaged proteins to maintain physiological functions. We found that FtsH hydrolyzes the Salmonella MgtC virulence protein when we substitute the MgtC 226th Trp, which is well conserved in other intracellular pathogens and normally protects MgtC from the FtsH-mediated proteolysis. Here we investigate a rule determining the FtsH-mediated proteolysis of the MgtC protein at Trp226 residue. Substitution of MgtC tryptophan 226th residue to alanine, glycine, or tyrosine leads to MgtC proteolysis in a manner dependent on the FtsH protease whereas substitution to phenylalanine, methionine, isoleucine, leucine, or valine resists MgtC degradation by FtsH. These data indicate that a large and hydrophobic side chain at 226th residue is required for protection from the FtsH-mediated MgtC proteolysis.

Peptide transporter YjiY influences the expression of the virulence gene mgtC to regulate biofilm formation in Salmonella.

Formation of a biofilm is one of the coping strategies of Salmonella against antimicrobial environmental stresses including nutrient starvation. However, the channeling of the starvation cue towards biofilm formation is not well understood. Our study shows that a carbon starvation gene, yjiY, coding for a peptide transporter, influences the expression of a virulence-associated gene mgtC in Salmonella to regulate biofilm formation. We demonstrate here that the mutant strain ΔyjiY is unable to form a biofilm due to the increased expression of mgtC. The upregulation of mgtC in the ΔyjiY strain correlates with the downregulation of the biofilm master regulator gene, csgD, and reduced levels of ATP. Our work further indicates that a yjiY-encoded peptide transporter may regulate the expression of mgtC by transporting proline peptides.

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.

Regulation and function of the Salmonella MgtC virulence protein.

Salmonella enterica serovar Typhimurium produces many virulence proteins to cause diseases. The Salmonella MgtC protein is one of such virulence proteins specially required for intracellular proliferation inside macrophages and mouse virulence. In this review, we will cover how the mgtC gene is turned on or off and what the signals required for mgtC expression are. Later in this review, we will discuss a recent understanding of MgtC function in Salmonella pathogenesis by identifying its target proteins.

A single amino acid of a Salmonella virulence protein contributes to pathogenicity by protecting from the FtsH-mediated proteolysis.

FtsH is a membrane-bound ATP-dependent protease in bacteria that is critical for degrading membrane proteins. The MgtC virulence protein from Salmonella enterica is located at the inner membrane and required for survival inside macrophages. Here we report that a single substitution at tryptophan 226 of the MgtC protein to alanine promotes the FtsH-mediated proteolysis. The Trp residue is located at the very C-terminus of the cytoplasmic domain of the MgtC protein and conserved only in intracellular pathogens surviving within a macrophage phagosome, suggesting that Salmonella may acquire the tryptophan residue to prevent MgtC degradation by the FtsH protease. Moreover, the reduced proteolytic activity of the FtsH protease during infection further increases MgtC production, promoting Salmonella's pathogenicity inside phagocytic cells.

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.