Shwu-Jen Liaw

Modulation of Swarming and Virulence by Fatty Acids through the RsbA Protein in Proteus mirabilis

ABSTRACT After sensing external signals, Proteus mirabilis undergoes a multicellular behavior called swarming which is coordinately regulated with the expression of virulence factors. Here we report that exogenously added fatty acids could act as signals to regulate swarming in P. mirabilis. Specifically, while oleic acid enhanced swarming, some saturated fatty acids, such as lauric acid, myristic acid, palmitic acid, and stearic acid, inhibited swarming. We also found that expression of hemolysin, which has been shown to be coordinately regulated with swarming, was also inhibited by the above saturated fatty acids. Previously we identified a gene, rsbA, which may encode a histidine-containing phosphotransmitter of the bacterial two-component signaling system and act as a repressor of swarming and virulence factor expression in P. mirabilis. We found that while myristic acid, lauric acid, and palmitic acid exerted their inhibitory effect on swarming and hemolysin expression through an RsbA-dependent pathway, the inhibition by stearic acid was mediated through an RsbA-independent pathway. Biofilm formation and extracellular polysaccharide (EPS) production play an important role in P. mirabilis infection. We found that RsbA may act as a positive regulator of biofilm formation and EPS production. Myristic acid was found to slightly stimulate biofilm formation and EPS production, and this stimulation was mediated through an RsbA-dependent pathway. Together, these data suggest that fatty acids may act as environmental cues to regulate swarming and virulence in P. mirabilis and that RsbA may play an important role in this process.

The RssAB Two-Component Signal Transduction System in Serratia marcescens Regulates Swarming Motility and Cell Envelope Architecture in Response to Exogenous Saturated Fatty Acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.

High Incidence of Cefoxitin and Clindamycin Resistance among Anaerobes in Taiwan

ABSTRACT Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The β-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For β-lactam-β-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-Bacteroidesfragilis species of the B. fragilis group were higher than those for B. fragilis (4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of B. fragilis group, Bacteroides thetaiotaomicron was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for B. fragilis continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of B. fragilis. We analyzed the genetic relationships among the metronidazole-resistant B. fragilis strains by pulsed-field gel electrophoresis. The metronidazole-resistant B. fragilis strains showed genotypic heterogeneity, excluding the dissemination of a single clone.

Multidrug resistance in clinical isolates of Stenotrophomonas maltophilia: roles of integrons, efflux pumps, phosphoglucomutase (SpgM), and melanin and biofilm formation

Integrons, efflux pumps, phosphoglucomutase (SpgM), and melanin and biofilm formation were investigated in 40 multidrug-resistant (MDR) and 30 non-MDR Stenotrophomonas maltophilia isolates recovered from patients treated at National Taiwan University Hospital (Taipei, Taiwan). Class 1 integrons were clearly associated with multidrug resistance. Sequencing data revealed that aminoglycoside-modifying genes occurred most frequently in the integron and disclosed a new bla(IMP-8)/aac6-II/aadA5 gene cassette. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed to assess the expression of Sme efflux pumps and SpgM in S. maltophilia. MDR isolates exhibited hyperexpression of SmeABC, and SmeDEF and SpgM were more frequently found in MDR isolates than non-MDR isolates. In addition, the ability of MDR isolates to form melanin-like pigment and biofilm was also greater than that of the non-MDR isolates. The SmeABC or SmeDEF pump was shown to be associated with resistance to all agents tested. The presence of an integron as well as production of pigment and biofilm was also responsible for resistance against eight, six and six of the tested agents, respectively. High SpgM expression was associated with resistance to only three of the tested agents. These findings define the important roles of integrons, efflux pumps, and melanin-like pigment and biofilm formation in the multidrug resistance of S. maltophilia. MDR isolates possessed more resistance mechanisms than susceptible strains.

Characterization of UDP-Glucose Dehydrogenase and UDP-Glucose Pyrophosphorylase Mutants of Proteus mirabilis: Defectiveness in Polymyxin B Resistance, Swarming, and Virulence

ABSTRACT Proteus mirabilis is known to be highly resistant to the action of polymyxin B (PB). However, the mechanism underlying PB resistance is not clear. In this study, we used Tn5 transposon mutagenesis to identify genes that may affect PB resistance in P. mirabilis. Two genes, ugd and galU, which may encode UDP-glucose dehydrogenase (Ugd) and UDP-glucose pyrophosphorylase (GalU), respectively, were identified. Knockout mutants of ugd and galU were found to be extremely sensitive to PB, presumably because of alterations in lipopolysaccharide (LPS) structure and cell surface architecture in these mutants. These mutants were defective in swarming, expressed lower levels of virulence factor hemolysin, and had lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd or galU gene, respectively, led to the restoration of wild-type phenotypic traits. Interestingly, we found that the expression of Ugd and GalU was induced by PB through RppA, a putative response regulator of the bacterial two-component system that we identified previously. Mutation in either ugd or galU led to activation of RpoE, an extracytoplasmic function sigma factor that has been shown to be activated by protein misfolding and alterations in cell surface structure in other bacteria. Activation of RpoE or RpoE overexpression was found to cause inhibition of FlhDC and hemolysin expression. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.

Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility

This study investigated the microbiological characteristics of 100 clinical isolates of Cryptococcus neoformans species complex, including serotypes, mating types, molecular types, antifungal susceptibility and virulence. The isolates were collected at National Taiwan University Hospital from 1999 to 2004. Eight isolates of C. neoformans from pigeon droppings were also evaluated. Among these isolates, 99 were C. neoformans var. grubii serotype A and one was C. neoformans var. gattii serotype B. All of these isolates were alpha mating types. PCR fingerprinting, generated by primers M13 and (GACA)(4), and URA5 gene restriction fragment length polymorphism analysis revealed that C. neoformans var. grubii isolates belonged to the VNI (98 isolates) and the VNII (one isolate) types, and the single C. neoformans var. gattii was VGI type. The similar profiles of clinical and environmental isolates suggest that patients might acquire these yeasts from the environment. The MIC(90) for fluconazole, itraconazole, 5-flucytosine, voriconazole and amphotericin B against all C. neoformans isolates were 8, 0.5, 4, 0.125 and 0.5 mg/L, respectively. All clinical isolates produced urease, phospholipase, capsule and melanin, but these activities varied with individual isolates. Analysis of six clinical and two environmental isolates with various levels of phospholipase activity indicated a correlation between phospholipase activity and the ability to adhere to the lung epithelial cell line, A549. The extent of cell damage, as indicated by lactate dehydrogenase release, also paralleled the phospholipase activity of these isolates. In addition, production of melanin contributed significant protection against amphotericin B killing of the isolates tested.

Characterisation of p-nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants.

p-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activities of swarming behaviour and virulence factor expression in Proteus mirabilis. The inhibitory action of PNPG was investigated by the isolation of Tn5 insertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super-swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the wild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor histidine kinase of the bacterial two-component signalling system. In the absence of PNPG, the mutants exhibited several swarming-related phenotypes that were different from those of the wild type; they initiated swarming earlier and had a less conspicuous consolidation phase, they differentiated earlier and maintained a differentiated state for longer, they started to express virulence factors earlier and maintained high expression levels of these factors for longer, and they had higher cell invasion ability than the wild type. These mutant phenotypes could be complemented by a plasmid-borne copy of rsbA. Together, these data suggest that RsbA may act as a repressor of swarming and virulence factor expression. In the presence of PNPG, these rsbA-mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, these data reveal a novel mechanism through which bacteria may negatively regulate swarming differentiation and virulence factor expression and identify a potential target of PNPG action.

Inhibition of virulence factor expression and swarming differentiation in Proteus mirabilis by p-nitrophenylglycerol

Proteus mirabilis is a common cause of upper urinary tract infections that can involve invasion of host urothelial cells. The ability to invade urothelial cells is coupled closely to swarming, a form of multicellular behaviour in which vegetative bacteria differentiate into hyperflagellate, filamentous swarming cells capable of co-ordinated and rapid population migration. Co-ordinate expression of virulence factors including urease, protease, haemolysin and flagellin during swarm-cell differentiation in P. mirabilis has been reported. To investigate the effects of p-nitrophenylglycerol (PNPG), a potent anti-swarming agent, on the various swarming-associated traits of P. mirabilis and to elucidate the relationships among them, P. mirabilis growth rate, swarming/swimming activity, cell invasion ability and the ability to express various virulence factors were monitored in the presence or absence of PNPG. It was found that PNPG could inhibit the growth rate, swarming differentiation and swarming/swimming activities of P. mirabilis. The expression of virulence factors such as protease, urease, haemolysin and flagellin in P. mirabilis was also inhibited by PNPG. The ability of P. mirabilis to invade human urothelial cells was reduced dramatically in the presence of PNPG. These results suggest that PNPG has the potential to be developed as an agent active against the effects of P. mirabilis infection.

Role of RppA in the Regulation of Polymyxin B Susceptibility, Swarming, and Virulence Factor Expression in Proteus mirabilis

ABSTRACT Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (>160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg2+. Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.

Proteus mirabilis pmrI, an RppA-Regulated Gene Necessary for Polymyxin B Resistance, Biofilm Formation, and Urothelial Cell Invasion

ABSTRACT Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.