bis(3--5-)-cyclic-diguanylic-acid and Gram-Negative-Bacterial-Infections

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium

Topics: Bacterial Physiological Phenomena; Caulobacter crescentus; Cell Cycle; Cyclic GMP; DNA Replication; Fimbriae, Bacterial; Gram-Negative Bacterial Infections; Models, Biological; Mutation

Our earlier studies showed that AhyRI- (AI-1) and LuxS-based (AI-2) quorum sensing (QS) systems were positive and negative regulators of virulence, respectively, in a diarrheal isolate SSU of Aeromonas hydrophila. Recently, we demonstrated that deletion of the QseBC two-component signal transduction system (AI-3 QS in enterohemorrhagic Escherichia coli) also led to an attenuation of A. hydrophila in a septicemic mouse model of infection, and that interplay exists between AI-1, AI-2, and the second-messenger cyclic-di-guanosine monophosphate (c-di-GMP) in modulating bacterial virulence. To further explore a network connection between all of the three QS systems in A. hydrophila SSU and their cross talk with c-di-GMP, we overproduced a protein with a GGDEF domain, which increases c-di-GMP levels in bacteria, and studied phenotypes and transcriptional profiling of genes involved in biofilm formation and motility of the wild-type (WT) A. hydrophila and its ΔqseB mutant. Over-expression of the GGDEF domain-encoding gene (aha0701h) resulted in a significantly reduced motility of the WT A. hydrophila similar to that of the ΔqseB mutant. While enhanced protease production was noted in WT A. hydrophila that had increased c-di-GMP, no enzymatic activity was detected in the ΔqseB mutant overexpressing the aha0701h gene. Likewise, denser biofilm formation was noted for WT bacteria when c-di-GMP was overproduced compared to its respective control; however, overproduction of c-di-GMP in the ΔqseB mutant led to reduced biofilm formation, a finding similar to that noted for the parental A. hydrophila strain. These effects on bacterial motility and biofilm formation in the ΔqseB mutant or the mutant with increased c-di-GMP were correlated with altered levels of fleN and vpsT genes. While we noted transcript levels of qseB and qseC genes to be increased in the ahyRI mutant, down-regulation of the ahyR and ahyI genes was observed in the ΔqseB mutant, which correlated with decreased protease activity. Finally, an enhanced virulence of WT A. hydrophila with increased c-di-GMP was noted in a mouse model when compared to findings in the parental strain with vector alone. Overall, we conclude that cross talk between AI-1 and QseBC systems exists in A. hydrophila SSU, and c-di-GMP modulation on QseBC system is dependent on the expression of the AI-1 system.

Topics: Aeromonas hydrophila; Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Female; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Mice; Quorum Sensing; Virulence

Francisella tularensis is a gram-negative bacterium that is highly virulent in humans, causing the disease tularemia. F. novicida is closely related to F. tularensis and exhibits high virulence in mice, but it is avirulent in healthy humans. An F. novicida-specific gene cluster (FTN0451 to FTN0456) encodes two proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains that modulate the synthesis and degradation of cyclic di-GMP (cdGMP). No DGC- or PDE-encoding protein genes are present in the F. tularensis genome. F. novicida strains lacking either the two DGC/PDE genes (cdgA and cdgB) or the entire gene cluster (strain KKF457) are defective for biofilm formation. In addition, expression of CdgB or a heterologous DGC in strain KKF457 stimulated F. novicida biofilms, even in a strain lacking the biofilm regulator QseB. Genetic evidence suggests that CdgA is predominantly a PDE, while CdgB is predominantly a DGC. The F. novicida qseB strain showed reduced cdgA and cdgB transcript levels, demonstrating an F. novicida biofilm signaling cascade that controls cdGMP levels. Interestingly, KKF457 with elevated cdGMP levels exhibited a decrease in intramacrophage replication and virulence in mice, as well as increased growth yields and biofilm formation in vitro. Microarray analyses revealed that cdGMP stimulated the transcription of a chitinase (ChiB) known to contribute to biofilm formation. Our results indicate that elevated cdGMP in F. novicida stimulates biofilm formation and inhibits virulence. We suggest that differences in human virulence between F. novicida and F. tularensis may be due in part to the absence of cdGMP signaling in F. tularensis.

Topics: Animals; Bacterial Proteins; Biofilms; Chitinases; Cyclic GMP; Female; Francisella; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Sequence Analysis, DNA; Virulence

Recently, we demonstrated that the LuxS-based quorum sensing (QS) system (AI-2) negatively regulated the virulence of a diarrheal isolate SSU of Aeromonas hydrophila, while the ahyRI-based (AI-1) N-acyl-homoserine lactone system was a positive regulator of bacterial virulence. Thus, these QS systems had opposing effects on modulating biofilm formation and bacterial motility in vitro models and in vivo virulence in a speticemic mouse model of infection. In this study, we linked these two QS systems with the bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) in the regulation of virulence in A. hydrophila SSU. To accomplish this, we examined the effect of overproducing a protein with GGDEF domain, which increases c-di-GMP levels in bacteria, on the phenotype and transcriptional profiling of genes involved in biofilm formation and bacterial motility in wild-type (WT) versus its QS null mutants. We provided evidence that c-di-GMP overproduction dramatically enhanced biofilm formation and reduced motility of the WT A. hydrophila SSU, which was equitable with that of the ΔluxS mutant. On the contrary, the ∆ahyRI mutant exhibited only a marginal increase in the biofilm formation with no effect on motility when c-di-GMP was overproduced. Overall, our data indicated that c-di-GMP overproduction modulated transcriptional levels of genes involved in biofilm formation and motility phenotype in A. hydrophila SSU in a QS-dependent manner, involving both AI-1 and AI-2 systems.

Topics: Aeromonas hydrophila; Biofilms; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Locomotion; Phenotype; Quorum Sensing; Transcription, Genetic; Virulence Factors