Role of the stress-responsive two-component system CpxAR in regulating fimbriae expression in Klebsiella pneumoniae CG43.

BACKGROUND: CpxAR is a two-component system that allows bacteria to reorganize envelope structures in response to extracellular stimuli. CpxAR negatively affects type 1 fimbriae expression in Klebsiella pneumoniae CG43, a hypervirulent strain. The involvement of CpxAR in the regulation of type 3 fimbriae expression was investigated. METHODS: cpxAR, cpxA, and cpxR gene-specific deletion mutants were generated. The deletion effects on the expression of type 1 and type 3 fimbriae were analyzed via measuring the promoter activity, mannose sensitive yeast agglutination activity, biofilm formation, and the production of the major pilins FimA and MrkA respectively. RNA sequencing analysis of CG43S3, ΔcpxAR, ΔcpxR and Δfur was employed to study the regulatory mechanism influencing the expression of type 3 fimbriae. RESULTS: Deletion of cpxAR increased type 1 and type 3 fimbrial expression. Comparative transcriptomic analysis showed that the expression of oxidative stress-responsive enzymes, type 1 and type 3 fimbriae, and iron acquisition and homeostasis control systems were differentially affected by cpxAR or cpxR deletion. Subsequent analysis revealed that the small RNA RyhB negatively affects the expression of type 3 fimbriae, while CpxAR positively controls ryhB expression. Finally, the site-directed mutation of the predicted interacting sequences of RyhB with the mRNA of MrkA attenuated the RyhB repression of type 3 fimbriae. CONCLUSION: CpxAR negatively regulates the expression of type 3 fimbriae by modulating cellular iron levels thereafter activating the expression of RyhB. The activated RyhB represses the expression of type 3 fimbriae by base-pairing binding to the 5'region of mrkA mRNA.

RcsB regulation of the YfdX-mediated acid stress response in Klebsiella pneumoniae CG43S3.

In Klebsiella pneumoniae CG43S3, deletion of the response regulator gene rcsB reduced the capsular polysaccharide amount and survival on exposure to acid stress. A comparison of the pH 4.4-induced proteomes between CG43S3 and CG43S3ΔrcsB revealed numerous differentially expressed proteins and one of them, YfdX, which has recently been reported as a periplasmic protein, was absent in CG43S3ΔrcsB. Acid survival analysis was then conducted to determine its role in the acid stress response. Deletion of yfdX increased the sensitivity of K. pneumoniae CG43S3 to a pH of 2.5, and transforming the mutant with a plasmid carrying yfdX restored the acid resistance (AR) levels. In addition, the effect of yfdX deletion was cross-complemented by the expression of the periplasmic chaperone HdeA. Furthermore, the purified recombinant protein YfdX reduced the acid-induced protein aggregation, suggesting that YfdX as well as HdeA functions as a chaperone. The following promoter activity measurement revealed that rcsB deletion reduced the expression of yfdX after the bacteria were subjected to pH 4.4 adaptation. Western blot analysis also revealed that YfdX production was inhibited by rcsB deletion and only the plasmid expressing RcsB or the nonphosphorylated form of RcsB, RcsBD56A, could restore the YfdX production, and the RcsB-mediated complementation was no longer observed when the sensor kinase RcsD gene was deleted. In conclusion, this is the first study demonstrating that YfdX may be involved in the acid stress response as a periplasmic chaperone and that RcsB positively regulates the acid stress response partly through activation of yfdX expression. Moreover, the phosphorylation status of RcsB may affect the YfdX expression under acidic conditions.

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43.

In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3ΔcpxAR, the derived mutants CG43S3ΔcpxARΔpecS and CG43S3ΔcpxARΔpecSΔpecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3ΔcpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3ΔcpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.

Oscillating gas bubbles as the origin of bowel sounds: a combined acoustic and imaging study.

Bowel sounds have been speculated to stem from the movement of gas or a mixture in the bowel lumen, with gas as the major component. The exact role and the mechanism through which gas participates have not been elucidated. Video images of actively moving bubbles under either real-time ultrasonography (RU, n = 4) or videofluoroscopy (VF, n = 4) with synchronous sound recording were studied and a total of 24 bubbling bowel sounds (BBS's) were obtained. The physical dimensions and acoustic parameters of bubbles were analyzed. Freely oscillating bubbles were demonstrated clearly in both groups. Bubble radii ranged from 1.5 to 7.2 mm and frequencies from 258.3 to 1,078 Hz. The bubble frequency correlated inversely with the radius (P < 0.01). The relevant acoustic features and parameters of bubble dynamics further supported the identification of gas bubbles. Although the acoustic features seemed to be of minor clinical significance, increased number of clustering or fixed, repetitive pattern of occurrences might suggest a poorer prognosis. In summary, oscillating gas bubbles are capable of producing BBS's and may play a central role in this newly recognized model of bowel sound genesis. The patterns of BBS's may be of prognostic value in clinical application, underlining the need for further study.

Ventricular hypertrophy and arterial hemodynamics following deprivation of nitric oxide in rats.

In the present study, we elucidated the possible role of hemodynamic parameters and chemical factors in the development of ventricular hypertrophy (VH) following chronic nitric oxide (NO) deprivation with Nomega-nitro-L-arginine methyl ester (L-NAME). Impedance spectral analysis was used to obtain the arterial hemodynamics including the steady and pulsatile components. Body weight (BW), left ventricular (LV) weight (LVW), LVW/BW ratio, LV collagen volume fraction (LVCVF), cyclic GMP, and nitrite/nitrate were measured. The extent of VH was evaluated by the LW/BW, total number, numerical density, and size of cardiomyocytes. Sprague-Dawley rats were given L-NAME 10, 20, and 40 mg/kg/day from the age of 10 to 18 weeks. Control and age-matched rats were given vehicle for the same period. Treatment of L-NAME for 8 weeks caused a dose-dependent increase in tail cuff pressure and a reduction in BW with increases in LVW, LVW/BW, number, numerical density, and size of myocytes. There was elevation of aortic pressure with decreases in cardiac output, and arterial compliance. The total peripheral resistance, characteristic impedance and pulse wave reflection were increased. Histological finding revealed severe myocardial hypertrophy and fibrosis with fibroblast infiltration. The LVCVF was increased, while LV cGMP and nitrite/nitrate were reduced in a dose-dependent manner. The results suggest that chronic NOS blockade causes hypertension, impairment of large vessel properties, and VH. The development of VH may result partly from the decreases in cGMP and nitrite/nitrate in the ventricle. Correlation analysis indicates that the extent of VH is equally related to the steady and pulsatile hemodynamics.