Rouh-Mei Hu

Induction of L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

ABSTRACT Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJΔL1 and KJΔL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.

Overexpression of Thy1/CD90 in human hepatocellular carcinoma is associated with HBV infection and poor prognosis.

Thy1/CD90 is an important marker of many types of stem cells. It functions as a tumor suppressor in ovarian cancer and in nasopharyngeal carcinoma. In this study, the expression status of Thy1 in clinical hepatocellular carcinoma (HCC) tissue samples was investigated. Relationships of Thy1 expression with clinical parameters and patient survival rate were analyzed. The quantities of Thy1 mRNA were statistically higher in tumor tissues than those in the adjacent non-tumor tissues (p<0.001). Immunohistochemical data confirmed that Thy1 protein was increased in 73% of HCC samples. Thy1 expression was not influenced by chronic alcohol exposure or cirrhosis. Overexpression in Thy1 was correlated with age (p=0.006), hepatitis B virus (HBV) infection (p=0.044), and histological grade (p=0.014). Patients with the highest level of Thy1 expression showed the poorest prognosis (p=0.040). In conclusion, overexpression of Thy1 may not suppress the development of HCC. Thy1 could provide a clinical prognostic marker for HCC.

AmpDI Is Involved in Expression of the Chromosomal L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

ABSTRACT Two ampD homologues, ampDI and ampDII, of Stenotrophomonas maltophilia have been cloned and analyzed. Comparative genomic analysis revealed that the genomic context of the ampDII genes is quite different, whereas that of the ampDI genes is more conserved in S. maltophilia strains. The ampD system of S. maltophilia is distinct from that of the Enterobacteriaceae and Pseudomonas aeruginosa in three respects. (i) AmpDI of S. maltophilia is not encoded in an ampDE operon, in contrast to what happens in the Enterobacteriaceae and P. aeruginosa. (ii) The AmpD systems of the Enterobacteriaceae and P. aeruginosa are generally involved in the regulation of ampR-linked ampC gene expression, while AmpDI of S. maltophilia is responsible for the regulation of two intrinsic β-lactamase genes, of which the L2 gene, but not the L1 gene, is linked to ampR. (iii) S. maltophilia exhibits a one-step L1 and L2 gene derepression model involving ampDI, distinct from the two- or three-step derepression of the Enterobacteriaceae and P. aeruginosa. Moreover, the ampDI and ampDII genes are constitutively expressed and not regulated by the inducer and AmpR protein, and the expression of ampDII is weaker than that of ampDI. Finally, AmpDII is not associated with the derepression of β-lactamases, and its role in S. maltophilia remains unclear.

The role of AmpR in regulation of L1 and L2 β-lactamases in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is known to produce at least two chromosomal-mediated inducible beta-lactamases, L1 and L2. Gene L2, which encodes a class A beta-lactamase, and the adjacent ampR gene form an ampR-class A beta-lactamase module. L1 belongs to the class B beta-lactamase and has no neighbor ampR-like regulatory gene. In this study, the ampR-L2 module from S. maltophilia KH was compared with ampR-beta-lactamase modules from several microorganisms with respect to the AmpR and beta-lactamase proteins and the intergenic (IG) region. S. maltophilia and Xanthomonas campestris showed the most closely phylogenetic relationship among the microorganisms considered. The regulatory role of AmpR towards L1 and L2 was further analyzed. In the absence of an inducer, AmpR acted as an activator for L1 expression and as a repressor for L2 expression, whereas AmpR was an activator for both genes in an induced state. In addition, inducibility of L1 and L2 genes depended on the presence of AmpR. The ampR transcript was weakly and constitutively expressed, but was not autoregulated.

An Inducible Fusaric Acid Tripartite Efflux Pump Contributes to the Fusaric Acid Resistance in Stenotrophomonas maltophilia

Background Fusaric acid (5-butylpicolinic acid), a mycotoxin, is noxious to some microorganisms. Stenotrophomonas maltophilia displays an intrinsic resistance to fusaric acid. This study aims to elucidate the mechanism responsible for the intrinsic fusaric acid resistance in S. maltophilia. Methodology A putative fusaric acid resistance-involved regulon fuaR-fuaABC was identified by the survey of the whole genome sequence of S. maltophilia K279a. The fuaABC operon was verified by reverse transcriptase-PCR. The contribution of the fuaABC operon to the antimicrobial resistance was evaluated by comparing the antimicrobials susceptibility between the wild-type strain and fuaABC knock-out mutant. The regulatory role of fuaR in the expression of the fuaABC operon was assessed by promoter transcription fusion assay. Results The fuaABC operon was inducibly expressed by fusaric acid and the inducibility was fuaR dependent. FuaR functioned as a repressor of the fuaABC operon in absence of a fusaric acid inducer and as an activator in its presence. Overexpression of the fuaABC operon contributed to the fusaric acid resistance. Significance A novel tripartite fusaric acid efflux pump, FuaABC, was identified in this study. Distinct from the formally classification, the FuaABC may constitute a new type of subfamily of the tripartite efflux pump.

SmeOP-TolCsm Efflux Pump Contributes to the Multidrug Resistance of Stenotrophomonas maltophilia

ABSTRACT A five-gene cluster, tolCSm-pcm-smeRo-smeO-smeP, of Stenotrophomonas maltophilia was characterized. The presence of smeOP and smeRo-pcm-tolCSm operons was verified by reverse transcription (RT)-PCR. Both operons were negatively regulated by the TetR-type transcriptional regulator SmeRo, as demonstrated by quantitative RT-PCR and a promoter-fusion assay. SmeO and SmeP were associated with TolCSm (the TolC protein of S. maltophilia) for the assembly of a resistance-nodulation-cell-division (RND)-type pump. The compounds extruded by SmeOP-TolCSm mainly included nalidixic acid, doxycycline, amikacin, gentamicin, erythromycin, leucomycin, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, sodium dodecyl sulfate, and tetrachlorosalicylanilide.

AmpN-AmpG Operon Is Essential for Expression of L1 and L2 β-Lactamases in Stenotrophomonas maltophilia

ABSTRACT AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter PampN. Expression from PampN was found to be independent of β-lactam exposure and ampN and ampG products. A ΔampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null β-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for β-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on β-lactamase expression. The AmpG permease of E. coli could complement the β-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for β-lactamase induction.

NagZ-Dependent and NagZ-Independent Mechanisms for β-Lactamase Expression in Stenotrophomonas maltophilia

ABSTRACT β-N-Acetylglucosaminidase (NagZ), encoded by the nagZ gene, is a critical enzyme for basal-level ampC derepression (ampC expression in the absence of β-lactam challenge) in ampD and dacB mutants of Pseudomonas aeruginosa. Three mutants with a phenotype of basal-level L1 and L2 β-lactamase derepression in Stenotrophomonas maltophilia have been reported, including KJΔDI (ampDI mutant), KJΔmrcA (mrcA mutant), and KJΔDIΔmrcA (ampDI and mrcA double mutant). In this study, nagZ of S. maltophilia was characterized, and its roles in basal-level β-lactamase derepression, induced β-lactamase activities, and β-lactam resistance of KJΔDI, KJΔmrcA, and KJΔDIΔmrcA were evaluated. Expression of the nagZ gene was constitutive and not regulated by AmpR, AmpDI, AmpN, AmpG, PBP1a, and NagZ. Introduction of ΔnagZ into KJΔDI nearly abolished basal-level derepressed β-lactamase activity; conversely, introduction of ΔnagZ into KJΔmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for β-lactamase expression in the S. maltophilia system, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed β-lactamase activities of KJΔDI and KJΔmrcA, respectively. The contributions of AL1 and AL2 to the induced β-lactamase activities may vary with the types of β-lactams. nagZ inactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced β-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced β-lactamase activities. Introduction of ΔnagZ into KJ, KJΔDI, KJΔmrcA, and KJΔDIΔmrcA did not significantly change the MICs of the β-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJΔZ and KJΔDIΔZ (nagZ mutants).

Role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia

OBJECTIVES To elucidate the role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia. METHODS The presence of the pcm-tolCsm operon was verified by RT-PCR. The phylogenetic relationship between the outer membrane proteins known to be involved in functional tripartite efflux in Escherichia coli, Pseudomonas aeruginosa and S. maltophilia was analysed. The contribution of TolCsm to resistance to a variety of compounds was investigated by susceptibility testing of the ΔtolCsm mutant. The role of pcm in the expression and function of tolCsm was assessed by quantitative real-time PCR and complementation assay. RESULTS The pcm and tolCsm genes formed an operon. TolCsm of S. maltophilia, OpmH of P. aeruginosa and TolC of E. coli formed a distinguishing phylogenetic TolC-like clade. TolCsm deletion increased the susceptibility of S. maltophilia KJ2 to several antimicrobial agents (aminoglycoside, macrolide, β-lactam, chloramphenicol, nalidixic acid, doxycycline and trimethoprim/sulfamethoxazole) and chemical compounds (acriflavine, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, fusaric acid, menadione, Paraquat, plumbagin, SDS and tetrachlorosalicylanilide). The in-frame deletion of pcm caused a polar effect on the expression of tolCsm, which compromised the resistance to amikacin and gentamicin. Nevertheless, the presence of the PCM protein made an insignificant contribution to the function of TolCsm in the resistance to amikacin and gentamicin. CONCLUSIONS The pcm-tolCsm operon makes a significant contribution to the multidrug resistance of S. maltophilia.

Characterization of a major facilitator superfamily (MFS) tripartite efflux pump EmrCABsm from Stenotrophomonas maltophilia

OBJECTIVES To characterize the emrRCABsm operon of Stenotrophomonas maltophilia. METHODS The presence of the emrRCABsm operon was verified by RT-PCR. The regulatory role of EmrRsm was investigated by ΔemrRsm mutant construction and promoter transcriptional fusion assay. A susceptibility test was employed to assess the substrate spectrum of the EmrCABsm efflux pump. The requirement for each component of the EmrCABsm pump was assessed by individual mutant construction and susceptibility testing. The expression of the emrRCABsm operon was evaluated by an induction assay, using different compounds as inducers. RESULTS emrRsm, emrCsm, emrAsm and emrBsm formed a four-member operon that was negatively regulated by the MarR-type transcriptional regulator EmrRsm. The emrRCABsm operon was intrinsically poorly expressed and the EmrCAB pump favoured extrusion of the uncoupling agents carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide (TCS), and the hydrophobic antibiotics nalidixic acid and erythromycin. However, the emrRCABsm operon could not be derepressed by CCCP, nalidixic acid, TCS, 2-chlorophenylhydrazine hydrochloride or salicylate, which are known to be possible inducers for MarR-type regulons. Each component of the EmrCABsm pump was apparently essential for pump function. CONCLUSIONS The EmrRsm-regulated EmrCABsm efflux pump is involved in the extrusion of hydrophobic compounds.