The involvement of PacIRA system of Stenotrophomonas maltophilia in the uptake of Pseudomonas aeruginosa pyochelin and intraspecies competition for iron acquisition.

BACKGROUND: Stenotrophomonas maltophilia, a species of highly genetic diversity, has emerged as an important nosocomial pathogen. S. maltophilia and Pseudomonas aeruginosa are often co-isolated from pneumonia patients. In our previous study, we have demonstrated that the pacIRA cluster present in some but not all clinical S. maltophilia isolates. Proteins encoded by pacIRA operon are an extracytoplasmic function (ECF) sigma factor, a transmembrane anti-sigma regulator, and a TonB-dependent receptor. This study aimed to elucidate PacIRA system function and its significance to S. maltophilia. METHODS: The pacI, pacR, and pacA genes were individually or totally deleted from the chromosome of KJΔEnt, a pacIRA-positive and siderophore-null strain. Growth promotion assay was performed to examine the implication of pacIRA system in iron utilization. Gene expression was quantified by quantitative real time PCR (qRT-PCR). Growth competition assay was executed to investigate the significance of pacIRA operon to S. maltophilia. RESULTS: PacIRA system contributed to utilize ferri-pyochelin of P. aeruginosa as iron sources for growth in an iron-depleted condition, but hardly utilized ferric citrate, hemin, ferri-stenobactin, and ferri-pyoverdine. PacIRA was founded to belong to Fur regulon and upregulated in response to iron-depleted stress. Growth competition assay demonstrated that pacIRA-positive S. maltophilia had a superiority over pacIRA-negative S. maltophilia in iron acquisition when they were co-cultured in P. aeruginosa ferri-pyochelin-supplemented medium. CONCLUSIONS: PacIRA system of S. maltophilia is a xenosiderophore uptake implement, involving in the acquisition of pyochelin of P. aeruginosa.

AmpR of Stenotrophomonas maltophilia is involved in stenobactin synthesis and enhanced ß-lactam resistance in an iron-depleted condition.

BACKGROUND: Iron is an essential nutrient for almost all aerobic organisms, including Stenotrophomonas maltophilia. Fur is the only known transcriptional regulator presumptively involved in iron homeostasis in S. maltophilia. AmpR, a LysR-type transcriptional regulator, is known to regulate ß-lactamase expression and ß-lactam resistance in S. maltophilia. OBJECTIVES: To identify the novel regulator involved in controlling the viability of S. maltophilia in an iron-depleted condition and to elucidate the underlying regulatory mechanisms. METHODS: The potential regulator involved in iron homeostasis was identified by studying the cell viabilities of different regulator mutants in 2,2'-dipyridyl (DIP)-containing medium. Iron-chelating activity was investigated using the chrome azurol S (CAS) activity assay. An iron source utilization bioassay was carried out to examine utilization of different iron sources. Gene expression was determined by quantitative real-time PCR, and the Etest method was used to evaluate antibiotic susceptibility. RESULTS: Of the 14 tested mutants, the ampR mutant, KJΔAmpR, showed a growth compromise in DIP-containing medium. AmpR regulated stenobactin synthesis in an iron-depleted condition, but showed little involvement in the uptake and utilization of ferri-stenobactin and ferric citrate. AmpR was up-regulated by iron limitation and ß-lactam challenge. S. maltophilia clinical isolates grown under conditions of iron depletion were generally more resistant to ß-lactams compared with conditions of iron repletion. CONCLUSIONS: AmpR is a dual transcriptional regulator in S. maltophilia, which regulates the ß-lactam-induced ß-lactamase expression and iron depletion-mediated stenobactin synthesis. AmpR is, therefore, a promising target for the development of inhibitors.

Roles of FadRACB system in formaldehyde detoxification, oxidative stress alleviation and antibiotic susceptibility in Stenotrophomonas maltophilia.

BACKGROUND: Formaldehyde toxicity is invariably stressful for microbes. Stenotrophomonas maltophilia, a human opportunistic pathogen, is widely distributed in different environments and has evolved an array of systems to alleviate various stresses. OBJECTIVES: To characterize the role of the formaldehyde detoxification system FadRACB of S. maltophilia in formaldehyde detoxification, oxidative stress alleviation and antibiotic susceptibility. METHODS: Presence of the fadRACB operon was verified by RT-PCR. Single or combined deletion mutants of the fadRACB operon were constructed for functional assays. Formaldehyde, menadione and quinolone susceptibilities were assessed by observing cell viability in formaldehyde-, menadione- and quinolone-containing media, respectively. Susceptibility to hydrogen peroxide was evaluated by disc diffusion assay. The agar dilution method was used to assess bacterial antibiotic susceptibilities. Expression of fadRACB was assessed by quantitative RT-PCR. RESULTS: The fadR, fadA, fadC and fadB genes were arranged in an operon. Mutants of fadA and/or fadB were more susceptible to formaldehyde and oxidative stress than the WT KJ strain of S. maltophilia. No significant difference was observed in the ability of a fadC single mutant to ameliorate formaldehyde and oxidative stress; however, simultaneous inactivation of fadA, fadB and fadC further enhanced susceptibility to formaldehyde and oxidative stress. In addition, compared with WT KJ, the triple mutant KJΔFadACB was more susceptible to quinolones and more resistant to aminoglycosides. FadR functions as a repressor for the fadRACB operon. The FadRACB operon has moderate expression in aerobically grown WT KJ and is further derepressed by formaldehyde challenge or oxidative stress, but not by antibiotics. CONCLUSIONS: The FadACB system contributes to mitigation of formaldehyde toxicity and oxidative stress and cross-protects S. maltophilia from quinolones.

Roles of the Two-MnSOD System of Stenotrophomonas maltophilia in the Alleviation of Superoxide Stress.

Manganese-dependent superoxide dismutase (MnSOD, SodA) and iron-dependent SOD (FeSOD, SodB) are critical cytosolic enzymes for alleviating superoxide stress. Distinct from the singular sodA gene in most bacteria, Stenotrophomonas maltophilia harbors two sodA genes, sodA1 and sodA2. The roles of SodA1, SodA2, and SodB of S. maltophilia in alleviating superoxide stress were investigated. The expression of sod genes was determined by promoter-xylE transcriptional fusion assay and qRT-PCR. SodA2 and sodB expressions were proportional to the bacterial logarithmic growth, but unaffected by menadione (MD), iron, or manganese challenges. SodA1 was intrinsically unexpressed and inducibly expressed by MD. Complementary expression of sodA1 was observed when sodA2 was inactivated. The individual or combined sod deletion mutants were constructed using the gene replacement strategy. The functions of SODs were assessed by evaluating cell viabilities of different sod mutants in MD, low iron-stressed, and/or low manganese-stressed conditions. Inactivation of SodA1 or SodA2 alone did not affect bacterial viability; however, simultaneously inactivating sodA1 and sodA2 significantly compromised bacterial viability in both aerobic growth and stressed conditions. SodA1 can either rescue or support SodA2 when SodA2 is defective or insufficiently potent. The presence of two MnSODs gives S. maltophilia an advantage against superoxide stress.