Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Type III Secretion System Gene Expression by Stimulating rsmYZ Transcription.

Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), which injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, we found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. We now demonstrate that mgtE expression acts through the GacAS two-component system to activate rsmY and rsmZ transcription. This event ultimately leads to inhibition of exsA translation. This inhibitory effect is specific to exsA as translation of other genes in the exsCEBA operon is not inhibited by mgtE Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli.IMPORTANCE The type III secretion system (T3SS) is a critical virulence factor utilized by numerous Gram-negative bacteria, including Pseudomonas aeruginosa, to intoxicate and kill host cells. Elucidating T3SS regulatory mechanisms may uncover targets for novel anti-P. aeruginosa therapeutics and provide deeper understanding of bacterial pathogenesis. We previously found that the magnesium transporter MgtE inhibits T3SS gene transcription in P. aeruginosa In this study, we describe the mechanism of MgtE-dependent inhibition of the T3SS. Our report also illustrates how MgtE might respond to environmental cues, such as magnesium levels, to fine-tune T3SS gene expression.

Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

DNA alternate polymerase PolB mediates inhibition of type III secretion in Pseudomonas aeruginosa.

Opportunistic pathogen Pseudomonas aeruginosa uses a variety of virulence factors to cause acute and chronic infections. We previously found that alternate DNA polymerase gene polB inhibits P. aeruginosa pyocyanin production. We investigated whether polB also affects T3SS expression. polB overexpression significantly reduced T3SS transcription and repressed translation of the master T3SS regulator ExsA, while not affecting exsA mRNA transcript abundance. Further, polB does not act through previously described genetic pathways that post-transcriptionally regulate ExsA. Our results show a novel T3SS regulatory component which may lead to development of future drugs to target this mechanism.

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces.

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.

RNA-Dependent Regulation of Virulence in Pathogenic Bacteria.

During infection, bacterial pathogens successfully sense, respond and adapt to a myriad of harsh environments presented by the mammalian host. This exquisite level of adaptation requires a robust modulation of their physiological and metabolic features. Additionally, virulence determinants, which include host invasion, colonization and survival despite the host's immune responses and antimicrobial therapy, must be optimally orchestrated by the pathogen at all times during infection. This can only be achieved by tight coordination of gene expression. A large body of evidence implicate the prolific roles played by bacterial regulatory RNAs in mediating gene expression both at the transcriptional and post-transcriptional levels. This review describes mechanistic and regulatory aspects of bacterial regulatory RNAs and highlights how these molecules increase virulence efficiency in human pathogens. As illustrative examples, Staphylococcus aureus, Listeria monocytogenes, the uropathogenic strain of Escherichia coli, Helicobacter pylori, and Pseudomonas aeruginosa have been selected.

Intestinal short-chain fatty acid composition does not explain gut microbiota-mediated effects on malaria severity.

Malaria is a devastating disease resulting in significant morbidity and mortality, especially in the developing world. Previously, we showed that the gut microbiome modulates severity of malaria in mice, though the exact mechanism was unknown. One well-studied mechanism by which the intestinal microbiota exerts an effect on host health is by synthesis of short-chain fatty acids (SCFAs). SCFAs have pleiotropic effects on the host, including modulating the immune system and altering susceptibility to pathogens. The objective of the current work was to explore if gut microbiota-mediated resistance and susceptibility to malaria in mice is through differential production of SCFAs. Of the eight detected SCFAs, only propionic acid (C3) was different between two groups of resistant and two groups of susceptible mice, with higher levels in feces of susceptible mice compared to resistant mice. Nevertheless, subsequent analysis revealed no robust correlation between malaria severity and levels of fecal propionic acid. In spite of the broad effect of SCFAs on host physiology, including host immunity, this study shows that gut microbiota-mediated modulation of malaria severity in mice is independent of fecal SCFA levels. Additionally, our data indicates that intestinal SCFAs do not function as biomarkers for prediction of malaria disease severity.

Correction: Intestinal short-chain fatty acid composition does not explain gut microbiota-mediated effects on malaria severity.

[This corrects the article DOI: 10.1371/journal.pone.0214449.].