Role of yceA-cybB-yceB operon in oxidative stress tolerance, swimming motility and antibiotic susceptibility of Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is ubiquitous in the environment and is an important MDR opportunistic pathogen. Oxidative stress is an inevitable challenge to an aerobic bacterium. Accordingly, S. maltophilia has many capabilities to face variable oxidative stress. Some of the oxidative stress alleviation systems cross-protect bacteria from antibiotics. In our recent RNA-sequencing transcriptome analysis, we documented the increased expression of a three-gene cluster, yceA-cybB-yceB, in the presence of hydrogen peroxide (H2O2). The YceI-like, cytochrome b561 and YceI-like proteins encoded by yceA, cybB and yceB are located in the cytoplasm, inner membrane and periplasm, respectively. OBJECTIVES: To characterize the role of the yceA-cybB-yceB operon of S. maltophilia in oxidative stress tolerance, swimming motility and antibiotic susceptibility. METHODS: The presence of the yceA-cybB-yceB operon was verified by RT-PCR. The functions of this operon were revealed by in-frame deletion mutant construction and complementation assay. Expression of the yceA-cybB-yceB operon was assessed by quantitative RT-PCR. RESULTS: The yceA, cybB and yceB genes form an operon. Loss of function of the yceA-cybB-yceB operon compromised menadione tolerance, enhanced swimming motility and increased susceptibility to fluoroquinolone and ß-lactam antibiotics. The expression of the yceA-cybB-yceB operon was up-regulated by oxidative stress, such as H2O2 and superoxide, and not impacted by antibiotics, such as fluoroquinolone and ß-lactams. CONCLUSIONS: The evidence strongly supports the view that the physiological function of the yceA-cybB-yceB operon is to alleviate oxidative stress. The operon provides an additional example that oxidative stress alleviation systems can cross-protect S. maltophilia from antibiotics.

Molecular Characterization of Three Tandemly Located Flagellin Genes of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a motile, opportunistic pathogen. The flagellum, which is involved in swimming, swarming, adhesion, and biofilm formation, is considered a virulence factor for motile pathogens. Three flagellin genes, fliC1, fliC2, and fliC3, were identified from the sequenced S. maltophilia genome. FliC1, fliC2, and fliC3 formed an operon, and their encoding proteins shared 67-82% identity. Members of the fliC1C2C3 operon were deleted individually or in combination to generate single mutants, double mutants, and a triple mutant. The contributions of the three flagellins to swimming, swarming, flagellum morphology, adhesion, and biofilm formation were assessed. The single mutants generally had a compromise in swimming and no significant defects in swarming, adhesion on biotic surfaces, and biofilm formation on abiotic surfaces. The double mutants displayed obvious defects in swimming and adhesion on abiotic and biotic surfaces. The flagellin-null mutant lost swimming ability and was compromised in adhesion and biofilm formation. All tested mutants demonstrated substantial but different flagellar morphologies, supporting that flagellin composition affects filament morphology. Bacterial swimming motility was significantly compromised under an oxidative stress condition, irrespective of flagellin composition. Collectively, the utilization of these three flagellins for filament assembly equips S. maltophilia with flagella adapted to provide better ability in swimming, adhesion, and biofilm formation for its pathogenesis.

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.

A sbiT-sbiRS-gloIo regulatory circuit is involved in oxidative stress tolerance of Stenotrophomonas maltophilia.

The sbiT-sbiR-sbiS operon of Stenotrophomonas maltophilia encodes an inner-membrane protein SbiT and a SbiS-SbiR two-component regulatory system. A sbiT mutant displayed a growth defect in LB agar. Mechanism studies revealed that sbiT deletion resulted in SbiSR activation and gloIo upregulation, which increased intracellular ROS level and caused growth defect.

Inpatient Outcomes of Patients Undergoing Robot-Assisted versus Laparoscopic Radical Cystectomy for Bladder Cancer: A National Inpatient Sample Database Study.

Background: Bladder cancer is a common urinary tract malignancy. Minimally invasive radical cystectomy has shown oncological outcomes comparable to the conventional open surgery and with advantages over the open procedure. However, outcomes of the two main minimally invasive procedures, robot-assisted and pure laparoscopic, have yet to be compared. This study aimed to compare in-hospital outcomes between these two techniques performed for patients with bladder cancer. Methods: This population-based, retrospective study included hospitalized patients aged ≥ 50 years with a primary diagnosis of bladder cancer who underwent robot-assisted or pure laparoscopic radical cystectomy. All patient data were extracted from the US National Inpatient Sample (NIS) database 2008-2018 and were analyzed retrospectively. Primary outcomes were in-hospital mortality, prolonged length of stay (LOS), and postoperative complications. Results: The data of 3284 inpatients (representing 16,288 US inpatients) were analyzed. After adjusting for confounders, multivariable analysis revealed that patients who underwent robot-assisted radical cystectomy had a significantly lower risk of in-hospital mortality (adjusted OR [aOR], 0.50, 95% CI: 0.28-0.90) and prolonged LOS (aOR, 0.63, 95% CI: 0.49-0.80) than those undergoing pure laparoscopic cystectomy. Patients who underwent robot-assisted radical cystectomy had a lower risk of postoperative complications (aOR, 0.69, 95% CI: 0.54-0.88), including bleeding (aOR, 0.73, 95% CI: 0.54-0.99), pneumonia (aOR, 0.49, 95% CI: 0.28-0.86), infection (aOR, 0.55, 95% CI: 0.36-0.85), wound complications (aOR, 0.33, 95% CI: 0.20-0.54), and sepsis (aOR, 0.49, 95% CI: 0.34-0.69) compared to those receiving pure laparoscopic radical cystectomy. Conclusions: Patients with bladder cancer, robot-assisted radical cystectomy is associated with a reduced risk of unfavorable short-term outcomes, including in-hospital mortality, prolonged LOS, and postoperative complications compared to pure laparoscopic radical cystectomy.

The sbiTRS Operon Contributes to Stenobactin-Mediated Iron Utilization in Stenotrophomonas maltophilia.

Iron is an essential micronutrient for various bacterial cellular processes. Fur is a global transcriptional regulator participating in iron homeostasis. Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has emerged as an opportunistic pathogen. To elucidate the novel regulatory mechanism behind iron homeostasis in S. maltophilia, wild-type KJ and KJΔFur, a fur mutant, were subjected to transcriptome assay. A five-gene cluster, sbiBA-sbiTRS, was significantly upregulated in KJΔFur. SbiAB is an ATP type efflux pump, SbiT is an inner membrane protein, and SbiSR is a two-component regulatory system (TCS). The sbiTRS operon organization was verified by reverse transcription-PCR (RT-PCR). Localization prediction and bacterial two-hybrid studies revealed that SbiT resided in the inner membrane and had an intramembrane interaction with SbiS. In iron-replete conditions, SbiT interacted with SbiS and maintained SbiSR TCS in a resting state. In response to iron depletion stress, SbiT no longer interacted with SbiS, leading to SbiSR TCS activation. The iron source utilization assay demonstrated the contribution of SbiSR TCS to stenobactin-mediated ferric iron utilization but notto the utilization of hemin and ferric citrate. Furthermore, SmeDEF and SbiAB pumps, known stenobactin secretion outlets, were members of the SbiSR regulon. Collectively, in an iron-depleted condition, SbiSR activation is regulated by Fur at the transcriptional level and by SbiT at the posttranslational level. Activated SbiSR contributes to stenobactin-mediated ferric iron utilization by upregulating the smeDEF and sbiAB operons. SbiSR is the first TCS found to be involved in iron homeostasis in S. maltophilia. IMPORTANCE Therapeutic options for Stenotrophomonas maltophilia infections are limited because S. maltophilia is intrinsically resistant to several antibiotics. Iron is an essential element for viability, but iron overload is a lethal threat to bacteria. Therefore, disruption of iron homeostasis can be an alternative strategy to cope with S. maltophilia infection. The intricate regulatory networks involved in iron hemostasis have been reported in various pathogens; however, little is known about S. maltophilia. Herein, a novel sbiTRS operon, a member of Fur regulon, was characterized. SbiT, an inner membrane protein, negatively modulated the SbiSR two-component regulatory system by intramembrane protein-protein interaction with SbiS. In response to iron-depleted stress, SbiSR was activated via the regulation of Fur and SbiT. Activated SbiSR upregulated smeDEF and sbiAB, which contributed to stenobactin-mediated ferric iron utilization. A novel fur-sbiT-sbiSR-smeDEF/sbiAB regulatory circuit in S. maltophilia was revealed.

σP-NagA-L1/L2 Regulatory Circuit Involved in ΔompA299-356-Mediated Increase in ß-Lactam Susceptibility in Stenotrophomonas maltophilia.

OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of ß-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA299-356, an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJΔOmpA299-356 was more susceptible to ß-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA299-356-mediated increase in ß-lactam susceptibility (abbreviated as "ΔOmpA299-356 phenotype"). KJΔOmpA299-356 displayed a lower ceftazidime (CAZ)-induced ß-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 ß-lactamases-null mutant, and KJ2ΔOmpA299-356, a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable ß-lactam susceptibility. Both lines of evidence indicate that decreased ß-lactamase activity contributes to the ΔOmpA299-356 phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA299-356, focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA299-356. Deletion of the nagA gene from the chromosome of KJΔOmpA299-356 restored ß-lactam susceptibility and CAZ-induced ß-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA299-356 contributes to the ΔOmpA299-356 phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA299-356. The deletion mutant construction, ß-lactam susceptibility, and ß-lactamase activity analysis demonstrated that σP, but not σE, was involved in the ΔOmpA299-356 phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σP regulon. The involvement of the σP-NagA-L1/L2 regulatory circuit in the ΔOmpA299-356 phenotype was manifested. IMPORTANCE Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed. The linkage between porin deficiency and antibiotic resistance increase has been reported widely, with a rationale for blocking antibiotic influx. In this study, a link between porin defects and ß-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel σP-NagA-L1/L2 regulatory circuit is triggered due to the loss of the OmpA-PG interaction. This study extends the understanding on the porin defect and antibiotic susceptibility. Porin defects may cause opposite impacts on antibiotic susceptibility, which is dependent on the involvement of the defect. Blocking the porin channel role can increase antibiotic resistance; in contrast, the loss of porin structure role may increase antibiotic susceptibility.

Roles of SmeYZ, SbiAB, and SmeDEF Efflux Systems in Iron Homeostasis of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia, a nonfermenting Gram-negative rod, is frequently isolated from the environment and is emerging as a multidrug-resistant global opportunistic pathogen. S. maltophilia harbors eight RND-type efflux pumps that contribute to multidrug resistance and physiological functions. Among the eight efflux pumps, SmeYZ pump is constitutively highly expressed. In our previous study, we demonstrated that loss-of-function of the SmeYZ pump results in pleiotropic phenotypes, including abolished swimming motility, decreased secreted protease activity, and compromised tolerance to oxidative stress and antibiotics. In this study, we attempted to elucidate the underlying mechanisms responsible for ΔsmeYZ-mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that smeYZ mutant experienced an iron starvation response because the genes involved in the synthesis and uptake of stenobactin, the sole siderophore of S. maltophilia, were significantly upregulated. We further verified that smeYZ mutant had low intracellular iron levels via inductively coupled plasma mass spectrometry (ICP-MS). Also, KJΔYZ was more sensitive to 2,2'-dipyridyl (DIP), a ferrous iron chelator, in comparison with the wild type. The contribution of SmeYZ, SmeDEF, and SbiAB pumps to stenobactin secretion was suggested by qRT-PCR and further verified by Chrome Azurol S (CAS) activity, iron source utilization, and cell viability assays. We also demonstrated that loss-of-function of SmeYZ led to the compensatory upregulation of SbiAB and SmeDEF pumps for stenobactin secretion. The overexpression of the SbiAB pump resulted in a reduction in intracellular iron levels, which may be the key factor responsible for the ΔsmeYZ-mediated pleiotropic phenotypes, except for antibiotic extrusion. IMPORTANCE Efflux pumps display high efficiency of drug extrusion, which underlies their roles in multidrug resistance. In addition, efflux pumps have physiological functions, and their expression is tightly regulated by various environmental and physiological signals. Functional redundancy of efflux pumps is commonly observed, and mutual regulation occurs among these functionally redundant pumps in a bacterium. Stenotrophomonas maltophilia is an opportunistic pathogen that shows intrinsic multi-drug resistance. In this study, we demonstrated that SmeYZ, SbiAB, and SmeDEF efflux pumps of S. maltophilia display functional redundancy in siderophore secretion. Inactivation of smeYZ led to the upregulation of smeDEF and sbiAB. Unexpectedly, sbiAB overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in smeYZ mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.

Involvement of the hemP-hemA-smlt0796-smlt0797 Operon in Hemin Acquisition by Stenotrophomonas maltophilia.

The hemin acquisition system of Stenotrophomonas maltophilia was elucidated in this study. To identify the TonB-dependent outer membrane receptor for hemin in S. maltophilia, the hemin acquisition systems of Pseudomonas aeruginosa were referenced. PhuR, HasA, and HxuA are three known TonB-dependent outer membrane receptors involved in hemin acquisition by P. aeruginosa. Thus, HemA (Smlt0795) and Smlt2937, the orthologs of PhuR and HasA/HxuA in S. maltophilia, were first considered. KJΔEnt, a stenobactin-null strain, was used as the parental strain for the hemin utilization assay. Deletion of hemA, but not Smlt2937, of KJΔEnt impaired hemin acquisition under iron-depleted conditions, indicating that HemA is the TonB-dependent receptor for hemin uptake. The hemA gene is a member of the hemP-hemA-smlt0796-smlt0797 operon, whose expression was upregulated in a fur mutant and under iron-depleted conditions. The contribution of the hemP-hemA-smlt0796-smlt0797 operon to hemin acquisition was investigated by in-frame deletion mutant construction and hemin utilization assays. Inactivation of hemP, smlt0796, and smlt0797 of KJΔEnt insignificantly affected hemin acquisition under iron-depleted conditions. However, hemP deletion in a fur mutant increased hemin acquisition under iron-depleted conditions. Collectively, we revealed that (i) HemA likely functions as the outer membrane receptor for hemin uptake; (ii) HemP, a predicted transcriptional factor, apparently functions as a repressor of the expression of the hemA transcript; and (iii) in a fur mutant, HemP has a negative impact on hemin acquisition under iron-depleted conditions. IMPORTANCE Stenotrophomonas maltophilia is an emerging multidrug-resistant opportunistic pathogen, increasing the difficulty of treatment of this infection. Iron is a critical element for bacterial viability. Heme is the most abundant iron source in the human host; thus, heme is the major iron source for a pathogen in the infection niche. Blocking iron acquisition from heme can be an alternative strategy to control S. maltophilia infection. Although several hemin acquisition systems have been reported in various pathogens, very little is known about the hemin acquisition systems of S. maltophilia. By in-frame deletion mutant construction and hemin utilization assays, we demonstrated that HemA (Smlt0795) is the TonB-dependent outer membrane receptor for hemin uptake and that HemP (Smlt0794), a predicted transcriptional factor, had a negative impact on hemin acquisition in a fur mutant. The negative regulatory role of HemP in hemin acquisition is first reported.