The bacillithiol pathway is required for biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus.

Diverse roles of low-molecular weight thiol GSH in Francisella's virulence, location sensing and GSH-stealing from host.

Low-molecular weight (LMW) thiols, encompassing peptides and small proteins with active cysteine residue(s), are important to bacteria as they are involved in a wide range of redox reactions. They include the tripeptide glutathione (GSH) and the small redox proteins, thioredoxins and glutaredoxins. We review the low MW thiols and related molecules in Francisella species and what role they may play in growth and virulence. Genes for GSH biosynthesis, metabolism and thioredoxins are present in all strains of Francisella, including the fully human-virulent strains. GSH and cysteine (CSH) are the major LMW thiols in Francisella extracts. We explore the potential role of the LMW thiols to overcome the nutritional challenges of intracellular growth (high GSH conditions) as well as the nutritional challenges of planktonic growth (low GSH conditions), and their contribution to Francisella's sensing its environmental location. Francisella may also use GSH as a source of CSH, for which it is auxotrophic. "Glutathione stealing" from the host may be an important part of Francisella's success strategy as a facultative intracellular pathogen both to detect its location and obtain CSH. An understanding of GSH metabolism in Francisella provides insights into the interaction of this pathogen with its host and may reveal additional targets for therapeutic intervention for tularemia infections.

Redox and Thiols in Archaea.

Low molecular weight (LMW) thiols have many functions in bacteria and eukarya, ranging from redox homeostasis to acting as cofactors in numerous reactions, including detoxification of xenobiotic compounds. The LMW thiol, glutathione (GSH), is found in eukaryotes and many species of bacteria. Analogues of GSH include the structurally different LMW thiols: bacillithiol, mycothiol, ergothioneine, and coenzyme A. Many advances have been made in understanding the diverse and multiple functions of GSH and GSH analogues in bacteria but much less is known about distribution and functions of GSH and its analogues in archaea, which constitute the third domain of life, occupying many niches, including those in extreme environments. Archaea are able to use many energy sources and have many unique metabolic reactions and as a result are major contributors to geochemical cycles. As LMW thiols are major players in cells, this review explores the distribution of thiols and their biochemistry in archaea.

N-methyl-bacillithiol, a Novel Thiol from Anaerobic Bacteria.

J. Hiras, S. V. Sharma, V. Raman, R. A. J. Tinson, et al. (mBio 9:e01603-18, 2018, https://doi.org/10.1128/mBio.01603-18) report on the identification of a novel thiol, N-methyl-bacillithiol (N-Me-BSH), in the green sulfur bacterium Chlorobium tepidum In N-methyl-bacillithiol, the amine of the cysteine is methylated by a novel S-adenosylmethioneine transferase designated N-methyl-bacillithiol synthase A (NmbA). The Hiras et al. study is significant because it is the first report of the presence of N-Me-BSH in anaerobic bacteria.

YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus.

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

Pseudomonas aeruginosa gshA Mutant Is Defective in Biofilm Formation, Swarming, and Pyocyanin Production.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause severe opportunistic infections. The principal redox buffer employed by this organism is glutathione (GSH). To assess the role of GSH in the virulence of P. aeruginosa, a number of analyses were performed using a mutant strain deficient in gshA, which does not produce GSH. The mutant strain exhibited a growth delay in minimal medium compared to the wild-type strain. Furthermore, the gshA mutant was defective in biofilm and persister cell formation and in swimming and swarming motility and produced reduced levels of pyocyanin, a key virulence factor. Finally, the gshA mutant strain demonstrated increased sensitivity to methyl viologen (a redox cycling agent) as well as the thiol-reactive antibiotics fosfomycin and rifampin. Taken together, these data suggest a key role for GSH in the virulence of P. aeruginosaIMPORTANCEPseudomonas aeruginosa is a ubiquitous bacterium that can cause severe opportunistic infections, including many hospital-acquired infections. It is also a major cause of infections in patients with cystic fibrosis. P. aeruginosa is intrinsically resistant to a number of drugs and is capable of forming biofilms that are difficult to eradicate with antibiotics. The number of drug-resistant strains is also increasing, making treatment of P. aeruginosa infections very difficult. Thus, there is an urgent need to understand how P. aeruginosa causes disease in order to find novel ways to treat infections. We show that the principal redox buffer, glutathione (GSH), is involved in intrinsic resistance to the fosfomycin and rifampin antibiotics. We further demonstrate that GSH plays a role in P. aeruginosa disease and infection, since a mutant lacking GSH has less biofilm formation, is less able to swarm, and produces less pyocyanin, a pigment associated with infection.

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress.

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.

Genome Sequences of Two Spore-Forming Bacteria Isolated from the Shore of Mono Lake, California.

Here, we report the draft genome sequences of two Bacillus spore-forming Gram-positive bacteria, isolated from soil on the shore of Mono Lake.

Genome Sequences of Three Spore-Forming Bacteria Isolated from the Feces of Organically Raised Chickens.

Antibiotic feed supplements have been implicated in the rise of multidrug-resistant bacteria. An alternative to antibiotics is probiotics. Here, we report the genome sequences of two Bacillus and one Solibacillus species, all spore-forming, Gram-positive bacteria, isolated from the feces organically raised chicken feces, with potential to serve as probiotics.

S-nitrosomycothiol reductase and mycothiol are required for survival under aldehyde stress and biofilm formation in Mycobacterium smegmatis.

We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. © 2016 IUBMB Life, 68(8):621-628, 2016.

Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis.

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.

The synergetic effect of ambient PM2.5 exposure and rhinovirus infection in airway dysfunction in asthma: a pilot observational study from the Central Valley of California.

BACKGROUND: Elevated levels of particulate matter PM2.5 and rhinovirus infection have been known to exacerbate asthma. However, the combined effect of rhinovirus infection and high PM2.5 has not been investigated. PURPOSE: To investigate the effect of PM2.5 and concomitant rhinovirus infection on airway function in asthma in an area with high PM2.5 concentration. METHODS: Asthmatics and their matched controls were monitored for lung function, exhaled nitric oxide (eNO) and respiratory symptoms on days with varying levels of PM2.5. As the study was a repeated measure design, repeated clinical findings, and laboratory data were used in the mixed model analysis. RESULTS: Wheezing and dyspnea in asthmatics were worsened with increasing ambient PM2.5. Increasing PM2.5 decreased FEV1% predicted (-0.51, -0.79 to -0.23) and FEF25-75% predicted (-0.66, -1.07 to -0.24) in subjects with asthma (all P < .01). Rhino viral infection reduced FEF25-75% predicted in subjects with asthma (-11.7, -20 to -2.9). The reductions in FEV25-75 and FEV1 per 10 µg/m(3) increase in ambient PM2.5 were 6% and 5% respectively. A significant interaction was observed between presence of rhinovirus infection and elevated PM2.5 in asthmatics causing a 4-fold decrease in FEF25-75 (P = .01) and a 2-fold decrease in FEV1% predicted values (P = .01) compared with asthmatics with no rhino viral infection. CONCLUSIONS: Increasing ambient PM2.5 and low temperature independently worsened airway function in asthma. The interaction between rhinovirus and PM2.5 significantly impairs airway function in asthma. A larger sample size study is suggested to investigate these observations.

Analysis of mutants disrupted in bacillithiol metabolism in Staphylococcus aureus.

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low molecular weight thiol found in low GC Gram-positive bacteria, such as Staphylococcus aureus. Like other low molecular weight thiols, BSH is likely involved in protection against a number of stresses. We examined S. aureus transposon mutants disrupted in each of the three genes associated with BSH biosynthesis. These mutants are sensitive to alkylating stress, oxidative stress, and metal stress indicating that BSH and BSH-dependent enzymes are involved in protection of S. aureus. We further demonstrate that BshB, a deacetylase involved in the second step of BSH biosynthesis, also acts as a BSH conjugate amidase and identify S. aureus USA 300 LAC 2626 as a BSH-S-transferase, which is able to conjugate chlorodinitrobenzene, cerulenin, and rifamycin to BSH.

Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus.

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Mycoredoxin-1 is one of the missing links in the oxidative stress defence mechanism of Mycobacteria.

To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of -218 mV and hydrogen bonding with neighbouring residues lowers the pKa of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

Characterization of BshA, bacillithiol glycosyltransferase from Staphylococcus aureus and Bacillus subtilis.

The first step during bacillithiol (BSH) biosynthesis involves the formation of N-acetylglucosaminylmalate from UDP-N-acetylglucosamine and l-malate and is catalyzed by a GT4 class glycosyltransferase enzyme (BshA). Recombinant Staphylococcus aureus and Bacillus subtilis BshA were highly specific and active with l-malate but the former showed low activity with d-glyceric acid and the latter with d-malate. We show that BshA is inhibited by BSH and similarly that MshA (first enzyme of mycothiol biosynthesis) is inhibited by the final product MSH.

Detoxification of toxins by bacillithiol in Staphylococcus aureus.

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low-molecular-mass thiol found in bacteria such as Bacillus sp., Staphylococcus aureus and Deinococcus radiodurans. Like other low-molecular-mass thiols such as glutathione and mycothiol, BSH is likely to be involved in protection against environmental toxins including thiol-reactive antibiotics. We report here a BSH-dependent detoxification mechanism in S. aureus. When S. aureus Newman strain was treated with monobromobimane and monochlorobimane, the cellular BSH was converted to the fluorescent S-conjugate BS-bimane. A bacillithiol conjugate amidase activity acted upon the BS-bimane to produce Cys-bimane, which was then acetylated by an N-acetyltransferase to generate N-acetyl-Cys-bimane, a mercapturic acid. An S. aureus mutant lacking BSH did not produce mercapturic acid when treated with monobromobimane and monochlorobimane, confirming the involvement of bacillithiol. Furthermore, treatment of S. aureus Newman with rifamycin, the parent compound of the first-line anti-tuberculosis drug, rifampicin, indicated that this thiol-reactive antibiotic is also detoxified in a BSH-dependent manner, since mercapturic acids of rifamycin were observed in the culture medium. These data indicate that toxins and thiol-reactive antibiotics are detoxified to less potent mercapturic acids in a BSH-dependent manner and then exported out of the cell in S. aureus.

The DinB superfamily includes novel mycothiol, bacillithiol, and glutathione S-transferases.

The superfamily of glutathione S-transferases has been the subject of extensive study; however, Actinobacteria produce mycothiol (MSH) in place of glutathione, and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates, an MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning, and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily, but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887, but En. faecalis produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Because more than two-thirds of the sequences assigned to the DinB superfamily are members of these families, it seems likely that such activity is dominant in the DinB superfamily.

Organic hydroperoxide resistance protein and ergothioneine compensate for loss of mycothiol in Mycobacterium smegmatis mutants.

The mshA::Tn5 mutant of Mycobacterium smegmatis does not produce mycothiol (MSH) and was found to markedly overproduce both ergothioneine and an ~15-kDa protein determined to be organic hydroperoxide resistance protein (Ohr). An mshA(G32D) mutant lacking MSH overproduced ergothioneine but not Ohr. Comparison of the mutant phenotypes with those of the wild-type strain indicated the following: Ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional MSH-dependent organic hydroperoxide peroxidase exists; and elevated isoniazid resistance in the mutant is associated with both Ohr and the absence of MSH. Purified Ohr showed high activity with linoleic acid hydroperoxide, indicating lipid hydroperoxides as the likely physiologic targets. The reduction of oxidized Ohr by NADH was shown to be catalyzed by lipoamide dehydrogenase and either lipoamide or DlaT (SucB). Since free lipoamide and lipoic acid levels were shown to be undetectable in M. smegmatis, the bound lipoyl residues of DlaT are the likely source of the physiological dithiol reductant for Ohr. The pattern of occurrence of homologs of Ohr among bacteria suggests that the ohr gene has been distributed by lateral transfer. The finding of multiple Ohr homologs with various sequence identities in some bacterial genomes indicates that there may be multiple physiologic targets for Ohr proteins.