Alkyl Hydroperoxide Reductase Research Articles

Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis.

Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.

Proteomic insights into the response of Halomonas sp. MNB13 to excess Mn(Ⅱ) and the role of H2S in Mn(Ⅱ) resistance

Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H2S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H2S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H2O2 and O2•−. Upon exogenous addition of H2S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H2O2 and O2•− in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H2S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H2S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria.

Comparative Genomic and Transcriptomic Analysis of Phenol Degradation and Tolerance in Acinetobacter lwoffii through Adaptive Evolution.

Microorganism-based methods have been widely applied for the treatment of phenol-polluted environments. The previously isolated Acinetobacter lwoffii NL1 strain could completely degrade 0.5 g/L phenol within 12 h, but not higher concentrations of phenol. In this study, we developed an evolutionary strain NL115, through adaptive laboratory evolution, which possessed improved degradation ability and was able to degrade 1.5 g/L phenol within 12 h. Compared with that of the starting strain NL1, the concentration of degradable phenol by the developed strain increased three-fold; its phenol tolerance was also enhanced. Furthermore, comparative genomics showed that sense mutations mainly occurred in genes encoding alkyl hydroperoxide reductase, phenol hydroxylase, 30S ribosomal protein, and mercury resistance operon. Comparative transcriptomics between A. lwoffii NL115 and NL1 revealed the enrichment of direct degradation, stress resistance, and vital activity processes among the metabolic responses of A. lwoffii adapted to phenol stress. Among these, all the upregulated genes (log2fold-change > 5) encoded peroxidases. A phenotypic comparison of A. lwoffii NL1 and NL115 found that the adapted strain NL115 exhibited strengthened antioxidant capacity. Furthermore, the increased enzymatic activities of phenol hydroxylase and alkyl hydroperoxide reductase in A. lwoffii NL115 validated their response to phenol. Overall, this study provides insight into the mechanism of efficient phenol degradation through adaptive microbial evolution and can help to drive improvements in phenol bioremediation.

Ahp deficiency-induced redox imbalance leads to metabolic alterations in E.coli

Alkyl hydroperoxide reductase (Ahp) is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli (E. coli). Ahp-deficient strains have been found to have high reactive oxygen species (ROS) levels, sufficient to cause cell damage. However, the exact role and underlying mechanisms of Ahp deficiency-induced cell damage remain largely unknown. Here, the E. coli MG1655 ΔAhp mutant strain was constructed as a model of deficiency to assess its role. The cells of the ΔAhp strain were found to be significantly longer than those of the wild strain, with elevated ROS and hydrogen peroxide (H2O2) levels. Proteome, redox proteome and metabolome analyses were performed to systematically present a global and quantitative profile and delineate the redox signaling and metabolic alterations at the proteome, metabolome, and cysteine oxidation site levels. The multiomics data revealed that Ahp deficiency disrupted the redox balance, activated the OxyR system, upregulated oxidative defense proteins and inhibited the TCA cycle to some extent. Surprisingly, the mutant strain shifted from aerobic respiration to anaerobic respiration and fermentation during the logarithmic phase in the presence of sufficient O2. The acid resistance system was activated to mitigate the effect of excessive acid produced by fermentation. Taken together, the results of this study demonstrated that Ahp deficiency triggered cellular redox imbalance and regulated metabolic pathways to confer resistance to submicromolar intracellular H2O2 levels in E. coli.

Unraveling the Role of the Zinc-Dependent Metalloproteinase/HTH-Xre Toxin/Antitoxin (TA) System of Brucella abortus in the Oxidative Stress Response: Insights into the Stress Response and Virulence.

Toxin/antitoxin (TA) systems have been scarcely studied in Brucella abortus, the causative agent of brucellosis, which is one of the most prevalent zoonotic diseases worldwide. In this study, the roles of a putative type II TA system composed by a Zinc-dependent metalloproteinase (ZnMP) and a transcriptional regulator HTH-Xre were evaluated. The deletion of the open reading frame (ORF) BAB1_0270, coding for ZnMP, used to produce a mutant strain, allowed us to evaluate the survival and gene expression of B. abortus 2308 under oxidative conditions. Our results showed that the B. abortus mutant strain exhibited a significantly reduced capacity to survive under hydrogen peroxide-induced oxidative stress. Furthermore, this mutant strain showed a decreased expression of genes coding for catalase (katE), alkyl hydroperoxide reductase (ahpC) and transcriptional regulators (oxyR and oxyR-like), as well as genes involved in the general stress response, phyR and rpoE1, when compared to the wild-type strain. These findings suggest that this type II ZnMP/HTH-Xre TA system is required by B. abortus to resist oxidative stress. Additionally, previous evidence has demonstrated that this ZnMP also participates in the acidic stress resistance and virulence of B. abortus 2308. Therefore, we propose a hypothetical regulatory function for this ZnMP/HTH-Xre TA system, providing insight into the stress response and its potential roles in the pathogenesis of B. abortus.

Polystyrene microplastics biodegradation by gut bacterial Enterobacter hormaechei from mealworms under anaerobic conditions: Anaerobic oxidation and depolymerization

Synthetic plastic is used throughout daily life and industry, threatening organisms with microplastic pollution. Polystyrene is a major plastic polymer and also widely found sources of plastic wastes and microplastics. Here, we report that Enterobacter hormaechei LG3 (CP118279.1), a facultative anaerobic bacterial strain isolated from the gut of Tenebrio molitor larvae (mealworms) can oxidize and depolymerize polystyrene under anaerobic conditions. LG3 performed biodegradation while forming a biofilm on the plastic surface. PS biodegradation was characterized by analyses of surface oxidation, change in morphology and molecular weights, and production of biodegraded derivative. The biodegradation performance by LG3 was compared with PS biodegradation by Bacillus amyloliquefaciens SCGB1 under both anaerobic and aerobic conditions. In addition, through nanopore sequencing technology, we identified degradative enzymes, including thiol peroxidase (tpx), alkyl hydroperoxide reductase C (ahpC) and bacterioferritin comigratory protein (bcp). Along with the upregulation of degradative enzymes for biodegradation, changes in lipid A and biofilm-associated proteins were also observed after the cells were incubated with polystyrene microplastics. Our results provide evidence for anaerobic biodegradation by polystyrene-degrading bacteria and show alterations in gene expression patterns after polystyrene microplastics treatment in the opportunistic pathogen Enterobacter hormaechei.

Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition.

Multiple drug resistance (MDR) bacterial strains are responsible by 1.2 million of human deaths all over the world. The pathogens possess efficient enzymes which are able to mitigate the toxicity of reactive oxygen species (ROS) produced by some antibiotics and the host immune cells. Among them, the bacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is able to decompose efficiently several kinds of hydroperoxides. To decompose their substrates AhpC use a reactive cysteine residue (peroxidatic cysteine-CysP) that together with two other polar residues (Thr/Ser and Arg) comprise the catalytic triad of these enzymes and are involved in the substrate targeting/stabilization to allow a bimolecular nucleophilic substitution (SN2) reaction. Additionally to the high efficiency the AhpC is very abundant in the cells and present virulent properties in some bacterial species. Despite the importance of AhpC in bacteria, few studies aimed at using natural compounds as inhibitors of this class of enzymes. Some natural products were identified as human isoforms, presenting as common characteristics a bulk hydrophobic moiety and an α, β-unsaturated carbonylic system able to perform a thiol-Michael reaction. In this work, we evaluated two chemically related natural products: 1,4-dihydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzene (C1) and 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C2), both were isolated from branches Piper crassinervium (Piperaceae), over the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC). By biochemical assays we show that although both compounds can perform the Michael addition reaction, only compound C2 was able to inhibit the PaAhpC peroxidase activity but not SeAhpC, presenting IC50 = 20.3 μM. SDS-PAGE analysis revealed that the compound was not able to perform a thiol-Michael addition, suggesting another inhibition behavior. Using computer-assisted simulations, we also show that an acidic group present in the structure of compound C2 may be involved in the stabilization by polar interactions with the Thr and Arg residues from the catalytic triad and several apolar interactions with hydrophobic residues. Finally, C2 was not able to interfere in the peroxidase activity of the isoform Prx2 from humans or even the thiol proteins of the Trx reducing system from Escherichia coli (EcTrx and EcTrxR), indicating specificity for P. aeruginosa AhpC.

Mycobacterium abscessus alkyl hydroperoxide reductase C promotes cell invasion by binding to tetraspanin CD81

Mycobacterium abscessus (Mab) is an increasingly recognized pulmonary pathogen. How Mab is internalized by macrophages and establishes infection remains unknown. Here, we show that Mab uptake is significantly reduced in macrophages pre-incubated with neutralizing anti-CD81 antibodies or in cells in which the large extracellular loop (LEL) of CD81 has been deleted. Saturation of Mab with either soluble GST-CD81-LEL or CD81-LEL-derived peptides also diminished internalization of the bacilli. The mycobacterial alkyl hydroperoxide reductase C (AhpC) was unveiled as a major interactant of CD81-LEL. Pre-exposure of macrophages with soluble AhpC inhibited mycobacterial uptake whereas overexpression of AhpC in Mab enhanced its internalization. Importantly, pre-incubation of macrophages with anti-CD81-LEL antibodies inhibited phagocytosis of AhpC-coated beads, indicating that AhpC is a direct interactant of CD81-LEL. Conditional depletion of AhpC in Mab correlated with decreased internalization of Mab. These compelling data unravel a previously unexplored role for CD81/AhpC to promote uptake of pathogenic mycobacteria by host cells.

Metabolite profiling, in vitro and in silico assessment of antibacterial and anticancer activities of Alternaria alternata endophytic in Jatropha heynei.

Fungal endophytes produce a range of structurally diverse metabolites with bioactive principles. In this study, an endophytic fungus Alternaria alternata was isolated from Jatropha heynei and cultured in potato dextrose liquid broth. Culture filtrate of A. alternata was extracted in ethyl acetate and metabolites were characterized by QTOF-HRLCMS. Among compounds detected, spectral compounds such as kigelinone, and levofuraltadone were reported with antibacterial property, while 2-hydroxychrasophanol, isoathyriol, glycophymoline, columbianetin and kaempferol 3-O-β-D- galactoside were reported with cytotoxic properties. Partially purified metabolites of A. alternata showed significant antibacterial activity against tested clinical bacterial strains by agar well diffusion method. High zone of inhibition was recorded against Enterococcus faecalis, Pseudomonas syringae and Klebsiella pneumoniae. In vitro anticancer activity of fungal extract by MTT assay displayed high cytotoxic effect on human lung carcinoma cancer cell line (A549) with IC50 value of 393.52µgml-1, and without any significant cytotoxic effect on human breast cancer cell line (MCF-7). Further, antibacterial and anticancer spectral compounds of A. alternata were subjected to molecular docking analysis with antibacterial target proteins such as tellurite resistance protein (2JXU), indole-3-acetaldehyde dehydrogenase (5IUU) and alkyl hydroperoxide reductase (5Y63), and anticancer target human apoptotic regulator protein (1G5M). The results of the study indicated that kigelinone, levofuraltadone, 2-hydroxychrasophanol and isoathyriolin the fungal extract have significant binding modes, with best binding energy scores with their respective antibacterial and anticancer target proteins. Alternaria alternata resident in J. heynei offers a promising source of broad-spectrum antibacterial and anticancer compounds.

Indole and azaindole halogenation catalyzed by the RebH enzyme variant 3-LSR utilizing co-purified E. coli reductase.

Biocatalytic C-H halogenation is becoming increasingly attractive due to excellent catalyst-controlled selectivity and environmentally benign reaction conditions. Significant efforts have been made on enzymatic halogenation of industrial arenes in a cost-effective manner. Here we report an unprecedented enzymatic halogenation of a panel of industrially important indole, azaindole and anthranilamide derivatives using a thermostable RebH variant without addition of any external flavin reductase enzyme. The reactions were catalyzed by the RebH variant 3-LSR enzyme with the help of a co-purified E. coli reductase identified as alkyl hydroperoxide reductase F (AhpF).

Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up

Backgroundα-Ionone is highly valued in cosmetics and perfumery with a global usage of 100–1000 tons per year. Metabolic engineering by microbial fermentation offers a promising way to produce natural (R)-α-ionone in a cost-effective manner. Apart from optimizing the metabolic pathways, the approach is also highly dependent on generating a robust strain which retains productivity during the scale-up process. To our knowledge, no study has investigated strain robustness while increasing α-ionone yield.ResultsBuilt on our previous work, here, we further increased α-ionone yield to 11.4 mg/L/OD in 1 mL tubes by overexpressing the bottleneck dioxygenase CCD1 and re-engineering the pathway, which is > 65% enhancement as compared to our previously best strain. However, the yield decreased greatly to 2.4 mg/L/OD when tested in 10 mL flasks. Further investigation uncovered an unexpected inhibition that excessive overexpression of CCD1 was accompanied with increased hydrogen peroxide (H2O2) production. Excessive H2O2 broke down lycopene, the precursor to α-ionone, leading to the decrease in α-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of α-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved α-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H2O2. The strain exhibited improved robustness and produced ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature.ConclusionOur study provides an insight on the importance of mediating the oxidative stress to improve strain robustness and microbial production of α-ionone during scaling up. This new strategy may be inspiring to the biosynthesis of other high-value apocarotenoids such as retinol and crocin, in which oxygenases are also involved.

Clinical Utility of Combined Whole-cell Antigen and Recombinant Hemolysis Co-regulated Protein 1-Enzyme-linked Immunosorbent Assays Reveals Underdiagnosed Cases of Melioidosis in Vietnam.

Melioidosis is a fatal infectious disease in the tropics and subtropics. Currently, bacterial culture is the gold standard for diagnosis of the disease, but its sensitivity is relatively low. In this study, we evaluated four ELISAs using sera collected from culture-confirmed cases of melioidosis (n = 63), cases with other bacterial infections (n = 62), and healthy donors (n = 60). Antigens used for ELISAs were the whole-cell (WC) antigens and recombinant proteins of hemolysis co-regulated protein 1 (Hcp1), GroEL1, and alkyl hydroperoxide reductase subunit C (AhpC). Using the cutoff values for optical density at 490 nm defined at a specificity of > 95%, the sensitivity of the WC, Hcp1, GroEL1, and AhpC ELISAs was 93.7%, 87.3%, 61.9%, and 57.1%, respectively. The combined WC/Hcp1 ELISA showed the greatest sensitivity and specificity of 98.4% and 95.1%, respectively. Of 511 and 500 sera collected from clinically suspected febrile patients admitted to the General Hospital of Ha Tinh Province and the Hue Central Hospital, respectively, combined WC/Hcp1 ELISAs showed 52 (10.2%) and 41 (8.2%) patients positive for melioidosis, respectively. The assay detected 14 of 14 (100%) and 21 of 23 (91.3%) culture-confirmed cases of melioidosis at Ha Tinh and Hue, respectively. A follow-up study of 38 patients positive for melioidosis by combined WC/Hcp1 ELISAs but negative for Burkholderia pseudomallei by culture method or not assigned to examine for bacterial culture resulted in 2 (5.3%) culture-reconfirmed patients with melioidosis, 9 (23.7%) deaths, 17 (44.7%) unhealthy patients, and 10 (26.3%) healthy persons. Combined WC/Hcp1 ELISA was a reliable serological method to detect underdiagnosed cases of melioidosis. Further investigations are needed to estimate the true sensitivity and specificity of the assay and the true number of cases of melioidosis.

Development of Fluoroquinolone Resistance through Antibiotic Tolerance in Campylobacter jejuni.

ABSTRACTAntibiotic tolerance not only enables bacteria to survive acute antibiotic exposures but also provides bacteria with a window of time in which to develop antibiotic resistance. The increasing prevalence of Campylobacter jejuni isolates resistant to clinically important antibiotics, particularly fluoroquinolones (FQs), is a global public health concern. Currently, little is known about antibiotic tolerance and its effects on resistance development in C. jejuni. Here, we show that exposure to ciprofloxacin or tetracycline at concentrations 10 and 100 times higher than the MIC induces antibiotic tolerance in C. jejuni, whereas gentamicin or erythromycin treatment causes cell death. Interestingly, FQ resistance rapidly develops in C. jejuni after tolerance induction by ciprofloxacin and tetracycline. Furthermore, after tolerance is induced, alkyl hydroperoxide reductase (AhpC) plays a critical role in reducing FQ resistance development by alleviating oxidative stress. Together, these results demonstrate that exposure of C. jejuni to antibiotics can induce antibiotic tolerance and that FQ-resistant (FQR) C. jejuni clones rapidly emerge after tolerance induction. This study elucidates the mechanisms underlying the high prevalence of FQR C. jejuni and provides insights into the effects of antibiotic tolerance on resistance development.IMPORTANCE Antibiotic tolerance compromises the efficacy of antibiotic treatment by extending bacterial survival and facilitating the development of mutations associated with antibiotic resistance. Despite growing public health concerns about antibiotic resistance in C. jejuni, antibiotic tolerance has not yet been investigated in this important zoonotic pathogen. Here, our results show that exposure of C. jejuni to ciprofloxacin or tetracycline leads to antibiotic tolerance development, which subsequently facilitates the emergence of FQR C. jejuni. Importantly, these antibiotics are commonly used in animal agriculture. Moreover, our study suggests that the use of non-FQ drugs in animal agriculture promotes FQ resistance development, which is crucial because antibiotic-resistant C. jejuni is primarily transmitted from animals to humans. Overall, these findings increase our understanding of the mechanisms of resistance development through the induction of antibiotic tolerance.

The enigmatic physiological roles of AhpCF, Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H2O2) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H2O2 which avoids accumulation of the peroxide to lethal concentrations.

Transcriptome analysis reveals manganese tolerance mechanisms in a novel native bacterium of Bacillus altitudinis strain HM-12

Bacillus altitudinis HM-12, isolated from ferromanganese ore tailings, can resist up to 1200 mM Mn(II) when exposed to concentrations from 50 mM to 1400 mM. HM-12 exhibited high Mn(II) removal efficiency (90.6 %). We report the transcriptional profile of HM-12 using RNA-Seq and found 423 upregulated and 536 downregulated differentially expressed genes (DEGs) compared to the control. Gene Ontology analysis showed that DEGs were mainly linked with transporter activity, binding, catalytic activity in molecular function, cellular anatomical entity in cellular component, cellular process, and metabolic process. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEGs were mostly mapped to membrane transport, signal transduction, carbohydrate and amino acid metabolism, energy metabolism, and cellular community pathways. Transport analysis showed that two manganese importer systems, mntH and mntABC, were significantly downregulated. The manganese efflux genes (mneS, yceF and ykoY) exhibited significant upregulation. Manganese homeostasis seems to be subtly regulated by manganese uptake and efflux genes. Moreover, it was found that copA as a Mn(II) oxidase gene and a copper chaperone gene copZ were considerably upregulated by signal transduction analysis. csoR encoding a transcriptional repressor which can regulate the copZA operon was upregulated. The strong Mn(II) oxidizing activity of HM-12 was also confirmed by physicochemical characterization. In metabolism and environmental information processing, yjqC encoding manganese catalase was significantly upregulated, while katE and katX encoding heme catalases were significantly downregulated. The antioxidant gene pcaC was significantly upregulated, but ykuU encoding alkyl hydroperoxide reductase, yojM encoding superoxide dismutase, and perR encoding redox-sensing transcriptional repressor were downregulated. These results highlight the oxidative activity of HM-12 by regulating the transcription of oxidase, catalase, peroxidase, and superoxide dismutase to sense the cellular redox status and prevent Mn(II) intoxication. This study provides relevant information on the biological tolerance and oxidation mechanisms in response to Mn(II) stress.

Role of a Putative Alkylhydroperoxidase Rv2159c in the Oxidative Stress Response and Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is one of the leading infectious agents worldwide with a high rate of mortality. Following aerosol inhalation, M. tuberculosis primarily infects the alveolar macrophages, which results in a host immune response that gradually activates various antimicrobial mechanisms, including the production of reactive oxygen species (ROS), within the phagocytes to neutralize the bacteria. OxyR is the master regulator of oxidative stress response in several bacterial species. However, due to the absence of a functional oxyR locus in M. tuberculosis, the peroxidase stress is controlled by alkylhydroperoxidases. M. tuberculosis expresses alkylhydroperoxide reductase to counteract the toxic effects of ROS. In the current study, we report the functional characterization of an orthologue of alkylhydroperoxidase family member, Rv2159c, a conserved protein with putative peroxidase activity, during stress response and virulence of M. tuberculosis. We generated a gene knockout mutant of M. tuberculosis Rv2159c (MtbΔ2159) by specialized transduction. The MtbΔ2159 was sensitive to oxidative stress and exposure to toxic transition metals. In a human monocyte (THP-1) cell infection model, MtbΔ2159 showed reduced uptake and intracellular survival and increased expression of pro-inflammatory molecules, including IL-1β, IP-10, and MIP-1α, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Similarly, in a guinea pig model of pulmonary infection, MtbΔ2159 displayed growth attenuation in the lungs, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Our study suggests that Rv2159c has a significant role in maintaining the cellular homeostasis during stress and virulence of M. tuberculosis.

Dihydrolipoamide Acetyltransferase AceF Influences the Type III Secretion System and Resistance to Oxidative Stresses through RsmY/Z in Pseudomonas aeruginosa.

Carbon metabolism plays an important role in bacterial physiology and pathogenesis. The type III secretion system (T3SS) of Pseudomonas aeruginosa is a virulence factor that contributes to acute infections. It has been demonstrated that bacterial metabolism affects the T3SS. Meanwhile, expression of T3SS genes is negatively regulated by the small RNAs RsmY and RsmZ. In this study, we studied the relationship between the dihydrolipoamide acetyltransferase gene aceF and the T3SS. Our results reveal an upregulation of RsmY and RsmZ in the aceF mutant, which represses the expression of the T3SS genes. Meanwhile, the aceF mutant is more tolerant to hydrogen peroxide. We demonstrate that the expression levels of the catalase KatB and the alkyl hydroperoxide reductase AhpB are increased in the aceF mutant. The simultaneous deletion of rsmY and rsmZ in the aceF mutant restored the expression levels of katB and ahpB, as well as bacterial susceptibility to hydrogen peroxide. Thus, we identify a novel role of AceF in the virulence and oxidative response of P. aeruginosa.

Glutaredoxin Interacts with GR and AhpC to Enhance Low-Temperature Tolerance of Antarctic Psychrophile Psychrobacter sp. ANT206.

Glutaredoxin (Grx) is an important oxidoreductase to maintain the redox homoeostasis of cells. In our previous study, cold-adapted Grx from Psychrobacter sp. ANT206 (PsGrx) has been characterized. Here, we constructed an in-frame deletion mutant of psgrx (Δpsgrx). Mutant Δpsgrx was more sensitive to low temperature, demonstrating that psgrx was conducive to the growth of ANT206. Mutant Δpsgrx also had more malondialdehyde (MDA) and protein carbonylation content, suggesting that PsGrx could play a part in the regulation of tolerance against low temperature. A yeast two-hybrid system was adopted to screen interacting proteins of 26 components. Furthermore, two target proteins, glutathione reductase (GR) and alkyl hydroperoxide reductase subunit C (AhpC), were regulated by PsGrx under low temperature, and the interactions were confirmed via bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). Moreover, PsGrx could enhance GR activity. trxR expression in Δpsgrx, Δahpc, and ANT206 were illustrated 3.7, 2.4, and 10-fold more than mutant Δpsgrx Δahpc, indicating that PsGrx might increase the expression of trxR by interacting with AhpC. In conclusion, PsGrx may participate in glutathione metabolism and ROS-scavenging by regulating GR and AhpC to protect the growth of ANT206. These findings preliminarily suggest the role of PsGrx in the regulation of oxidative stress, which could improve the low-temperature tolerance of ANT206.

Mycobacterium avium Subsp. hominissuis Interactions with Macrophage Killing Mechanisms.

Non-tuberculosis mycobacteria (NTM) are ubiquitously found throughout the environment. NTM can cause respiratory infections in individuals with underlying lung conditions when inhaled, or systemic infections when ingested by patients with impaired immune systems. Current therapies can be ineffective at treating NTM respiratory infections, even after a long course or with multidrug treatment regimens. NTM, such as Mycobacterium avium subspecies hominissuis (M. avium), is an opportunistic pathogen that shares environments with ubiquitous free-living amoeba and other environmental hosts, possibly their evolutionary hosts. It is highly likely that interactions between M. avium and free-living amoeba have provided selective pressure on the bacteria to acquire survival mechanisms, which are also used against predation by macrophages. In macrophages, M. avium resides inside phagosomes and has been shown to exit it to infect other cells. M. avium’s adaptation to the hostile intra-phagosomal environment is due to many virulence mechanisms. M. avium is able to switch the phenotype of the macrophage to be anti-inflammatory (M2). Here, we have focused on and discussed the bacterial defense mechanisms associated with the intra-phagosome phase of infection. M. avium possesses a plethora of antioxidant enzymes, including the superoxide dismutases, catalase and alkyl hydroperoxide reductase. When these defenses fail or are overtaken by robust oxidative burst, many other enzymes exist to repair damage incurred on M. avium proteins, including thioredoxin/thioredoxin reductase. Finally, M. avium has several oxidant sensors that induce transcription of antioxidant enzymes, oxidation repair enzymes and biofilm- promoting genes. These expressions induce physiological changes that allow M. avium to survive in the face of leukocyte-generated oxidative stress. We will discuss the strategies used by M. avium to infect human macrophages that evolved during its evolution from free-living amoeba. The more insight we gain about M. avium’s mode of pathogenicity, the more targets we can have to direct new anti-virulence therapies toward.

Organic Hydroperoxide Resistance Gene ohr (VPA1681) Confers Protection against Organic Peroxides in the Presence of Alkyl Hydroperoxide Reductase Genes in Vibrio parahaemolyticus.

The marine foodborne enteropathogen Vibrio parahaemolyticus contains the chief organic peroxide reductases AphC1-AhpC2 and a putative organic hydroperoxide resistance enzyme (Ohr; VPA1681) against different peroxides. This study investigated the function of the Ohr under the presence of AhpC1-AhpC2 in this pathogen by gene mutation. Experimental results demonstrated that the ohr gene product was a weak scavenger of H2O2 only in the mutant strains that lacked the peroxide sensor/regulator oxyR and ahpC1-ahpC2 genes. The Ohr of V. parahaemolyticus was highly effective at scavenging organic peroxide, as demonstrated by assaying the defective changes in the Δohr mutant strain and determining the detoxifying activity of the purified recombinant V. parahaemolyticus Ohrvp protein in the reduced form. The Ohr and AhpC1-AhpC2 exhibited similar functions against organic peroxides; however, only the ΔahpC1ΔahpC2 mutant strain showed a significant increase in susceptibility to several disinfectants, organic acids, and antibiotics compared with the wild-type strain. The transcription of the ohr gene depended on exogenous cumene hydroperoxide (cumene) stress and was markedly enhanced in the ΔohrR (VPA1682) mutant strains. This study revealed the organic hydroperoxide reductase activity of the Ohr in V. parahaemolyticus, and its role probably depends on sophisticated regulation by OhrR. IMPORTANCE Vibrio parahaemolyticus is the most prevalent foodborne pathogen in Taiwan and some other coastal Asian countries, and its antioxidative activity contributes to the tolerance of this bacterium to different environmental stresses. This study reports on the function of the organic hydroperoxide resistance gene (ohr; VPA1681) and its gene regulator, ohrR (VPA1682), in this pathogen. The strain with the ohr gene had effective protection against organic peroxide, and the recombinant Ohrvp was active in its reduced form. The function of Ohr was significant mostly in strains in which the function of AhpC1-AhpC2 was limited. The ohrR repressor of the ohr gene was effective at low concentrations of organic peroxide. Other common Vibrio species that contain homologous ohr, ohrR, ahpC1, and ahpC2 genes, which are phylogenetically close to those of V. parahaemolyticus, may share similar functions to those revealed in this study.