Characterisation of the regulatory RNA RsmB from Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the molecular regulation of virulence factors and secondary metabolites is tightly controlled. This control involves several signal-mediated regulatory networks, including the GacS-GacA system and quorum sensing. Recently, the posttranscriptional repressor protein RsmA has been implicated in secondary metabolite production. RsmA is postulated to work in tandem with an as yet unidentified regulatory RNA molecule in a manner analogous to its homologues in other bacteria. Here we have identified a gene encoding an untranslated regulatory RNA (RsmB), located in the rpoS/ fdxA intergenic region of the P. aeruginosa PAO1 genome. Overexpression of rsmB in P. aeruginosa resulted in an increase in N-acyl-homoserine lactone, pyocyanin and elastase production compared with a marked decrease when rsmA was overexpressed. Mutation of rsmB resulted in a decrease in AHL production compared to wild type. We propose that RsmB is the cognate regulatory RNA of RsmA in P. aeruginosa. The global regulator GacA was not absolutely required for rsmB transcription in P. aeruginosa, as is the case in Pseudomonas fluorescens. However, GacA influenced the kinetics of rsmB transcription in that in late stationary phase the gacA mutant showed a substantial reduction in rsmB transcript levels compared to wild type. RsmA also influenced rsmB; in an rsmA mutant, the steady state level of rsmB transcript was reduced and this was due to a decrease in the transcription of rsmB. A balance in the levels of RsmA and RsmB may be an autoregulatory mechanism to ensure that RsmA is tightly controlled, as might be expected for such a potent global repressor.

α-2,3-sialyltransferase expression level impacts the kinetics of lipooligosaccharide sialylation, complement resistance, and the ability of Neisseria gonorrhoeae to colonize the murine genital tract.

UNLABELLED: Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis. IMPORTANCE: Neisseria gonorrhoeae has become resistant to nearly every therapeutic antibiotic used and is listed as an "urgent threat" by the Centers for Disease Control and Prevention. Novel therapies are needed to combat drug-resistant N. gonorrhoeae. Gonococci express an α-2,3-sialyltransferase (Lst) that can scavenge sialic acid from the host and use it to modify lipooligosaccharide (LOS). Sialylation of gonococcal LOS converts serum-sensitive strains to serum resistance, decreases antibody binding, and combats killing by neutrophils and antimicrobial peptides. Mutant N. gonorrhoeae that lack Lst (cannot sialylate LOS) are attenuated in a mouse model. Lst expression levels differ among N. gonorrhoeae strains, and N. gonorrhoeae typically expresses more Lst than Neisseria meningitidis. Here we examined the significance of differential lst expression levels and determined that the level of LOS sialylation is critical to the ability of N. gonorrhoeae to combat the immune system and survive in an animal model. LOS sialylation may be an ideal target for novel therapies.

The post-transcriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts.

The importance of Csr post-transcriptional systems is gradually emerging; these systems control a variety of virulence-linked physiological traits in many pathogenic bacteria. This review focuses on the central role that Csr systems play in the pathogenesis of certain bacteria and in the establishment of successful infections in animal hosts. Csr systems appear to control the 'switch' between different physiological states in the infection process; for example switching pathogens from a colonization state to a persistence state. Csr systems are controlled by two-component sensor/regulator systems and by non-coding RNAs. In addition, recent findings suggest that the RNA chaperone Hfq may play an integral role in Csr-mediated bacterial adaptation to the host environment.

Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis.

RsmA is a posttranscriptional regulatory protein in Pseudomonas aeruginosa that works in tandem with a small non-coding regulatory RNA molecule, RsmB (RsmZ), to regulate the expression of several virulence-related genes, including the N-acyl-homoserine lactone synthase genes lasI and rhlI, and the hydrogen cyanide and rhamnolipid biosynthetic operons. Although these targets of direct RsmA regulation have been identified, the full impact of RsmA on cellular activities is not as yet understood. To address this issue the transcriptome profiles of P. aeruginosa PAO1 and an isogenic rsmA mutant were compared. Loss of RsmA altered the expression of genes involved in a variety of pathways and systems important for virulence, including iron acquisition, biosynthesis of the Pseudomonas quinolone signal (PQS), the formation of multidrug efflux pumps, and motility. Not all of these effects can be explained through the established regulatory roles of RsmA. This study thus provides both a first step towards the identification of further genes under RsmA posttranscriptional control in P. aeruginosa and a fuller understanding of the broader impact of RsmA on cellular functions.

Modulation of quorum sensing in Pseudomonas aeruginosa through alteration of membrane properties.

Changes in the cellular envelope are major physiological adaptations that occur when micro-organisms encounter extreme environmental conditions. An appropriate degree of membrane fluidity is crucial for survival, and alteration of membrane lipids is an essential adaptive response. Emerging data suggest that microbial cells may recognize alterations in their membrane viscosity resulting from certain environmental changes as a trigger for adaptive cellular responses. In Pseudomonas aeruginosa, the quorum-sensing (QS) system involves a complex regulatory circuitry that coordinates the expression of genes according to a critical population density. Interestingly, it has been shown that the QS system of P. aeruginosa can also be activated by nutritional stress, independently of the cell density, and therefore may be part of a more general adaptive response to stressful environmental conditions. In order to examine the proposed link between membrane properties and stress signalling, the effects of genetically engineered alterations of the membrane phospholipid composition of P. aeruginosa PAO1 on the activation of the stringent response and the QS system were examined. The lptA gene encoding a functional homologue of PlsC, an Escherichia coli enzyme that catalyses the second step of the phospholipid biosynthesis pathway, was identified and disrupted. Inactivation of lptA altered the fatty acid profile of phospholipids and the membrane properties, resulting in decreased membrane fluidity. This resulted in a premature production of the QS signals N-butanoyl- and N-hexanoyl-homoserine lactone (C4-HSL and C6-HSL) and a repression of 2-heptyl-3-hydroxy-4-quinolone (PQS) synthesis at later growth phases. The effects on C4- and C6-HSL depended upon the expression of relA, encoding the (p)ppGpp alarmone synthase, which was increased in the lptA mutant. Together, the findings support the concept that alterations in membrane properties can act as a trigger for stress-related gene expression.