Pseudomonas aeruginosa persister cell formation upon antibiotic exposure in planktonic and biofilm state.

Persister cell (PC) is dormant, tolerant to antibiotics, and a transient reversible phenotype. These phenotypes are observed in P. aeruginosa and cause bacterial chronic infection as well as recurrence of biofilm-mediated infection. PC formation requires stringent response and toxin-antitoxin (TA) modules. This study shows the P. aeruginosa PC formation in planktonic and biofilm stages on ceftazidime, gentamicin, and ciprofloxacin treatments. The PC formation was studied using persister assay, flow cytometry using Redox Sensor Green, fluorescence as well as Confocal Laser Scanning Microscopy, and gene expression of stringent response and TA genes. In the planktonic stage, ceftazidime showed a high survival fraction, high redox activity, and elongation of cells was observed followed by ciprofloxacin and gentamicin treatment having redox activity and rod-shaped cells. The gene expression of stringent response and TA genes were upregulated on gentamicin followed by ceftazidime treatment and varied among the isolates. In the biofilm stage, gentamicin and ciprofloxacin showed the biphasic killing pattern, redox activity, gene expression level of stringent response and TA varied across the isolates. Ceftazidime treatment showed higher persister cells in planktonic growth while all three antibiotics were able to induce persister cell formation in the biofilm stage.

The small RNA SprX regulates the autolysin regulator WalR in Staphylococcus aureus.

Pathogenesis of Staphylococcus aureus is attributed to its remarkable adaptation to changes in the environment, mediated by the arsenal of virulence factors, which are regulated by intricate mechanisms that include small RNAs (sRNAs) as important regulatory molecules. The sRNA SprX was previously described to be involved in the regulation of S. aureus pathogenicity, by modifying the expression of surface-associated clumping factor B and the secreted delta haemolysin. This study describes the regulation by SprX, of expression of multiple autolysins, which play an essential role in cell wall metabolism and function as important virulence factors that facilitate adhesion, internalization, and immune evasion during S. aureus colonization and pathogenesis. SprX acts by positively regulating the expression of autolysin regulator WalR. Overexpression of SprX resulted in differential regulation of autolysins IsaA, and LytM, while WalR levels were unchanged. SprX knockdown strain exhibited down-regulation of multiple autolytic bands corresponding to the major autolysin AtlA and its process intermediates in cell wall degradation zymography, and 0.2 to 0.1 fold reduction of lytM, atlA, isaA, and walR transcripts in qRT-PCRs. Down-regulation of SprX resulted in altered phenotype with high cell aggregation as analyzed by SEM, decrease in biofilm formation and higher resistance to Triton X-100-induced lysis, all of which indicate that SprX is essential for expression of autolysins. A putative RNA-RNA interaction was indicated in silico between SprX and walR mRNA and further confirmed by in vitro RNA-RNA interaction in electrophoretic mobility shift assays. These findings elucidate a new mechanism in which SprX modulates the S. aureus pathogenicity by regulating the regulator of autolysins in cell wall metabolism and as virulence factors.

Enhancement of the pathogenicity of Staphylococcus aureus strain Newman by a small noncoding RNA SprX1.

The pathogenesis of Staphylococcus aureus from local infection to systemic dissemination involves a range of virulence factors including structural and secreted products. Among various control mechanisms, small noncoding RNAs are involved in the regulation of multiple pathogenicity factors in S. aureus. The sRNA SprX which is encoded in the pathogenicity island of methicillin-susceptible S. aureus strain Newman and was shown to influence antibiotic resistance previously, upregulated the expression of virulence genes, especially the cell wall-associated clumping factor B (ClfB) and delta hemolysin (Hld). Bioinformatic analysis revealed several multiple mRNAs associated with pathogenicity as targets for SprX1, one of the three copies of sprX. Both overexpression and chromosomal disruption of sprX1 supported the scheme of upregulation of clfB and hld expression. Altered expression of SprX1 altered the levels of Hld and ClfB mRNAs, hemolysis, clumping of cells, biofilm formation by plate adhesion studies and confocal microscopic analysis as well as infection pathology of modified strains in mice models. ClfB and Hld mRNAs interacted directly with SprX1 in in vitro assays. Increased level of the regulatory RNA, namely RNAIII, that comprises Hld mRNA and also regulates the biofilm formation, indicates that SprX1 may also function through RNAIII for regulating virulence factors. An immunodominant protein, antigen A, was downregulated by SprX1 in two-dimensional electrophoresis. Taken together, these results signify the role of sRNA SprX in the pathogenicity of S. aureus Newman.