Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae . We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)- etcB (EIIB)- etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regulatory mechanism of etcABC in K. pneumoniae type 3 fimbriae formation was investigated. We found via quantitative RT-PCR that overexpression of etcABC enhanced the transcription level of the mrk operon, which is involved in type 3 fimbriae synthesis, and reduced the transcription level of the fim operon, which is involved in type 1 fimbriae synthesis. To gain further insight into the role of etcABC in type 3 fimbriae synthesis, we analyzed the region upstream of the mrk operon and found the potential cyclic 3′5′-adenosine monophosphate (cAMP) receptor protein (CRP) binding site. After crp was deleted in K. pneumoniae STU1 and two clinical isolates, these three crp mutant strains could not express MrkA, the major subunit of the fimbrial shaft, indicating that CRP positively regulated type 3 fimbriae synthesis. Moreover, a crp mutant overexpressing etcABC could not express MrkA, indicating that the regulation of type 3 fimbriae by etcABC was dependent on CRP. In addition, deletion of cyaA , which encodes the adenylyl cyclase that synthesizes cAMP, and deletion of crr , which encodes the glucose-specific EIIA, led to a reduction in lac operon regulation and therefore bacterial lactose uptake in K. pneumoniae . Exogenous cAMP but not etcABC overexpression compensated for the role of cyaA in bacterial lactose uptake. However, either etcABC overexpression or exogenous cAMP compensated for the role of crr in bacterial lac operon regulation that would eventually restore lactose uptake. We also found via ELISA and the luxCDABE reporter system that overexpression of etcABC increased intracellular cAMP levels and the transcription level of crp , respectively, in K. pneumoniae . In conclusion, overexpression of etcABC positively regulated cAMP production and cAMP-CRP activity to activate the mrk oper...
1,3-Propanediol (1,3-PD) is a valuable chemical intermediate in the synthesis of polyesters, polyethers, and polyurethanes, which have applications in various products such as cloth, bottles, films, tarpaulins, canoes, foam seals, high-resilience foam seating, and surface coatings. Klebsiella pneumoniae can produce 1,3-PD from glycerol. In this study, KPN00353, an EIIA homologue in the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), was found to play a negative regulatory role in 1,3-PD production under microaerobic conditions via binding to glycerol kinase (GlpK). The primary sequence of KPN00353 is similar to those of the fructose-mannitol EIIA (EIIFru and EIIAMtl) family. The interaction between KPN00353 and GlpK resulted in inhibition of the synthesis of glycerol-3-phosphate (G3P) and correlated with reductions in glycerol uptake and the production of 1,3-PD. Based on structure modeling, we conclude that residue H65 of KPN00353 plays an important role in the interaction with GlpK. We mutated this histidine residue to aspartate, glutamate, arginine and glutamine to assess the effects of each KPN00353 variant on the interaction with GlpK, on the synthesis of G3P and on the production of 1,3-PD. Our results illuminate the role of KPN00353 in 1,3-PD production by K. pneumoniae under microaerobic conditions.
Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.
The work performed by Ito et al was much appreciated since the authors addressed a novel and important issue in antibiotic rational uses, particularly the resistance of Cefmetazole in ESBL-producing E. coli and the involved gene regulations. Cefmetazole have been used as an alternative to carbapenems in addition to the use of quinolone and trimethoprim or sulfamethoxazole in infection cases caused by ESBL-producing E. coli. 1,2 In their study, 14 ESBLproducing and 12 ESBL-non-producing E. coli from 63 E. coli strains in total were isolated clinically. 3 The ESBLproducing ability of those E. coli isolates did not determine their behavior against Cefmetazole treatment in this study, since the MIC of either ESBL-non producing or ESBL-producing E. coli isolates vary (still relatively low at 1-4 μg/mL) in the first culture. Eleven strains of total 25 isolate gained resistance after being cultured with Cefmetazole at a low dose. Interestingly, after passage culture with the antibacterial-free medium, only 4 strains from these 11 isolates remain resistant to Cefmetazole, while the others gained susceptibility. The purpose of the authors was to unravel the mechanisms involved in the resistance acquisition against Cefmetazole in ESBL-producing E. coli clinical isolates, with the use of ESBL-non-producing isolates as the controls.The previous study, reported more than thirty years ago, addressed the potential mechanism of action of Cefmetazole in methicillin and cephem-resistant (MR) strains of Staphylococcus aureus. 4 However, the progression in this particular issue is relatively slower than the mechanism study of other type of antibiotic, especially in ESBL-producing E. coli. In this study, authors tried to explore the mechanism of how Cefmetazole resistance acquisition occurred in ESBLproducing E. coli isolates. Therefore, the transcription (mRNA) levels of porin encoding genes (ompF, ompC, phoE), chromosomal β-lactamase AmpC encoding genes (acrA, yhiV, mdfA), and drug efflux pump were detected in this particular study. However, as also discussed well in their discussion section, these mechanisms were not the novel part.Moreover, the authors also combined the use of Relebactam (the β-lactamase inhibitor) in observing the effects obtained in Cefmetazole use in E. coli isolates, which resulted in alteration in Cefmetazole susceptilbity. The addition of Relebactam suppressed the resistance toward Cefmetazole. However, the remaining question is, while β-lactamase was inhibited, what mechanism was underlying the suppression of Cefmetazole resistance acquisition? Is the effect caused by inhibiting β-lactamase alone enough to suppress antibiotic resistance? Should the upstream regulators be checked for details of their mechanisms, since the authors themselves mentioned that the Relebactam probably caused the porin deficiency. Novel regulation and details of the mechanisms involved probably could be unraveled if any transcription factors or specific motifs in DNA level were to be predicted and proven. 5,6 If there are any pred...
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