Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Antimicrobial Activity of Sertraline on Listeria monocytogenes.

We explored the antimicrobial activity of sertraline on Listeria monocytogenes and further investigated the effects of sertraline on biofilm formation and the virulence gene expression of L. monocytogenes. The minimum inhibitory concentration and minimum bactericidal concentration for sertraline against L. monocytogenes were in the range of 16-32 µg/mL and 64 µg/mL, respectively. Sertraline-dependent damage of the cell membrane and a decrease in intracellular ATP and pHin in L. monocytogenes were observed. In addition, sertraline reduced the biofilm formation efficiency of the L. monocytogenes strains. Importantly, low concentrations (0.1 µg/mL and 1 µg/mL) of sertraline significantly down-regulated the expression levels of various L. monocytogens virulence genes (prfA, actA, degU, flaA, sigB, ltrC and sufS). These results collectively suggest a role of sertraline for the control of L. monocytogenes in the food industry.

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements.

Zur and zinc increase expression of E. coli ribosomal protein L31 through RNA-mediated repression of the repressor L31p.

Bacteria can adapt in response to numerous stress conditions. One such stress condition is zinc depletion. The zinc-sensing transcription factor Zur regulates the way numerous bacterial species respond to severe changes in zinc availability. Under zinc sufficient conditions, Zn-loaded Zur (Zn2-Zur) is well-known to repress transcription of genes encoding zinc uptake transporters and paralogues of a few ribosomal proteins. Here, we report the discovery and mechanistic basis for the ability of Zur to up-regulate expression of the ribosomal protein L31 in response to zinc in E. coli. Through genetic mutations and reporter gene assays, we find that Zur achieves the up-regulation of L31 through a double repression cascade by which Zur first represses the transcription of L31p, a zinc-lacking paralogue of L31, which in turn represses the translation of L31. Mutational analyses show that translational repression by L31p requires an RNA hairpin structure within the l31 mRNA and involves the N-terminus of the L31p protein. This work uncovers a new genetic network that allows bacteria to respond to host-induced nutrient limiting conditions through a sophisticated ribosomal protein switching mechanism.

Agrochemical control of gene expression using evolved split RNA polymerase.

Chemically-inducible gene expression systems are valuable tools for rational control of gene expression both for basic research and biotechnology. However, most chemical inducers are confined to certain groups of organisms. Therefore, dissecting interactions between different organisms could be challenging using existing chemically-inducible systems. We engineered a mandipropamid-induced gene expression system (Mandi-T7) based on evolved split T7 RNAP system. As a proof-of-principle, we induced GFP expression in E. coli cells grown inside plant tissue.

An operator-based expression toolkit for Bacillus subtilis enables fine-tuning of gene expression and biosynthetic pathway regulation.

SignificanceA gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a malO operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of malO operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.

Cell density-dependent antibiotic tolerance to inhibition of the elongation machinery requires fully functional PBP1B.

The peptidoglycan (PG) cell wall provides shape and structure to most bacteria. There are two systems to build PG in rod shaped organisms: the elongasome and divisome, which are made up of many proteins including the essential MreB and PBP2, or FtsZ and PBP3, respectively. The elongasome is responsible for PG insertion during cell elongation, while the divisome is responsible for septal PG insertion during division. We found that the main elongasome proteins, MreB and PBP2, can be inhibited without affecting growth rate in a quorum sensing-independent density-dependent manner. Before cells reach a particular cell density, inhibition of the elongasome results in different physiological responses, including intracellular vesicle formation and an increase in cell size. This inhibition of MreB or PBP2 can be compensated for by the presence of the class A penicillin binding protein, PBP1B. Furthermore, we found this density-dependent growth resistance to be specific for elongasome inhibition and was consistent across multiple Gram-negative rods, providing new areas of research into antibiotic treatment.

Fosfomycin Resistance Evolutionary Pathways of Stenotrophomonas maltophilia in Different Growing Conditions.

The rise of multidrug-resistant Gram-negative pathogens and the lack of novel antibiotics to address this problem has led to the rescue of old antibiotics without a relevant use, such as fosfomycin. Stenotrophomonas maltophilia is a Gram-negative, non-fermenter opportunistic pathogen that presents a characteristic low susceptibility to several antibiotics of common use. Previous work has shown that while the so-far described mechanisms of fosfomycin resistance in most bacteria consist of the inactivation of the target or the transporters of this antibiotic, as well as the production of antibiotic-inactivating enzymes, these mechanisms are not selected in S. maltophilia fosfomycin-resistant mutants. In this microorganism, fosfomycin resistance is caused by the inactivation of enzymes belonging to its central carbon metabolism, hence linking metabolism with antibiotic resistance. Consequently, it is relevant to determine how different growing conditions, including urine and synthetic sputum medium that resemble infection, could impact the evolutionary pathways towards fosfomycin resistance in S. maltophilia. Our results show that S. maltophilia is able to acquire high-level fosfomycin resistance under all tested conditions. However, although some of the genetic changes leading to resistance are common, there are specific mutations that are selected under each of the tested conditions. These results indicate that the pathways of S. maltophilia evolution can vary depending on the infection point and provide information for understanding in more detail the routes of fosfomycin resistance evolution in S. maltophilia.

Insertion Sequence (IS) Element-Mediated Activating Mutations of the Cryptic Aromatic ß-Glucoside Utilization (BglGFB) Operon Are Promoted by the Anti-Terminator Protein (BglG) in Escherichia coli.

The cryptic ß-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic ß-glucoside. This may involve the insertion of an insertion sequence (IS) element into a "stress-induced DNA duplex destabilization" (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic ß-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG's binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.

Immunomodulatory Effects of Pure Cylindrospermopsin in Rats Orally Exposed for 28 Days.

Cylindrospermopsin (CYN) is a ubiquitous cyanotoxin showing increasing incidence worldwide. CYN has been classified as a cytotoxin and, among its toxic effects, its immunotoxicity is scarcely studied. This work investigates for the first time the influence of oral CYN exposure (18.75; 37.5 and 75 µg/kg b.w./day, for 28 days) on the mRNA expression of selected interleukin (IL) genes (IL-1ß, IL-2, IL-6, Tumor Necrosis Factor alpha (TNF-α), Interferon gamma (IFN-γ)) in the thymus and the spleen of male and female rats, by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, their serum levels were also measured by a multiplex-bead-based immunoassay, and a histopathological study was performed. CYN produced immunomodulation mainly in the thymus of rats exposed to 75 µg CYN/kg b.w./day in both sexes. However, in the spleen only IL-1ß and IL-2 (males), and TNF-α and IFN-γ (females) expression was modified after CYN exposure. Only female rats exposed to 18.75 µg CYN/kg b.w./day showed a significant decrease in TNF-α serum levels. There were no significant differences in the weight or histopathology in the organs studied. Further research is needed to obtain a deeper view of the molecular mechanisms involved in CYN immunotoxicity and its consequences on long-term exposures.

Halogenated Dihydropyrrol-2-One Molecules Inhibit Pyocyanin Biosynthesis by Blocking the Pseudomonas Quinolone Signaling System.

Quorum-sensing (QS) systems of Pseudomonas aeruginosa are involved in the control of biofilm formation and virulence factor production. The current study evaluated the ability of halogenated dihydropyrrol-2-ones (DHP) (Br (4a), Cl (4b), and F (4c)) and a non-halogenated version (4d) to inhibit the QS receptor proteins LasR and PqsR. The DHP molecules exhibited concentration-dependent inhibition of LasR and PqsR receptor proteins. For LasR, all compounds showed similar inhibition levels. However, compound 4a (Br) showed the highest decrease (two-fold) for PqsR, even at the lowest concentration (12.5 µg/mL). Inhibition of QS decreased pyocyanin production amongst P. aeruginosa PAO1, MH602, ATCC 25619, and two clinical isolates (DFU-53 and 364707). In the presence of DHP, P. aeruginosa ATCC 25619 showed the highest decrease in pyocyanin production, whereas clinical isolate DFU-53 showed the lowest decrease. All three halogenated DHPs also reduced biofilm formation by between 31 and 34%. The non-halogenated compound 4d exhibited complete inhibition of LasR and had some inhibition of PqsR, pyocyanin, and biofilm formation, but comparatively less than halogenated DHPs.

Drug Repurposing of the Unithiol: Inhibition of Metallo-ß-Lactamases for the Treatment of Carbapenem-Resistant Gram-Negative Bacterial Infections.

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

The Lipoteichoic Acid-Related Proteins YqgS and LafA Contribute to the Resistance of Listeria monocytogenes to Nisin.

Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A mariner transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants (ΔyqgS, ΔlafA, ΔvirR, ΔgtcA, and Δlmo1464) in L. monocytogenes. The results revealed that yqgS and lafA, the lipoteichoic acid-related genes, were essential for resistance to nisin, while the gtcA and lmo1464 mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on ΔyqgS and ΔlafA mutants under nisin treatment. Deletion of yqgS and lafA altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens. IMPORTANCE Listeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells.

The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa, the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa, which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Sub-Inhibitory Concentrations of Oxacillin, but Not Clindamycin, Linezolid, or Tigecycline, Decrease Staphylococcal Phenol-Soluble Modulin Expression in Community-Acquired Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus (SA) is a major human pathogen producing virulence factors, such as Panton-Valentine-leucocidin (PVL), alpha-hemolysin (Hla), and phenol-soluble-modulins alpha (PSMα), including delta-hemolysin (Hld). Unlike oxacillin, clindamycin and linezolid subinhibitory concentrations (sub-MIC) display an anti-toxin effect on PVL and Hla expression. Few studies have investigated PSMα and Hld expression modulation by antibiotics. Herein, we assessed the effect of antibiotic sub-MIC on PSMα1 and Hld expression for 4 community-acquired methicillin-resistant SA (CA-MRSA), 2 strains belonging to USASA300 and 2 strains belonging to ST80 European clone. SA were grown under oxacillin, clindamycin, linezolid, or tigecycline. After incubation, culture pellets were used for the determination of psmα1, pmtB, pmtR mRNA, and RNAIII levels by relative quantitative RT-PCR. PSMα1 and Hld expressions were measured in supernatant using high-performance-liquid-chromatography coupled to mass-spectrometry (HPLC-MS). Oxacillin sub-MIC reduced PSMα1 and Hld production, partially related to mRNA variations. For other antibiotics, effects on toxin expression were strain or clone dependent. Antibiotic effect on mRNA did not always reflect protein expression modulation. Variations of pmtB, pmtR mRNA, and RNAIII levels were insufficient to explain toxin expression modulation. Altogether, these data indicate that PSMα and Hld expressions are modulated by antibiotics (potential anti-toxin effect of oxacillin) differently compared to PVL and Hla. IMPORTANCE Staphylococcal toxins play an important role in the physiopathology of staphylococcal infections. Subinhibitory concentrations (sub-MIC) of antibiotics modulate in vitro toxins expression in S. aureus: clindamycin (CLI) and linezolid (LIN) display an anti-toxin effect on Panton-Valentine leucocidin and alpha-hemolysin production, while oxacillin (OXA) has an inducing effect. Few studies have focused on the modulation of phenol-soluble modulins alpha (PSMα) including delta-hemolysin expression by sub-MIC antibiotics. The aim of the present study was to investigate the effects of sub-MIC antibiotics on the expression of PSMα toxins for 4 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolates. The data presented herein confirm that OXA sub-MICs constantly inhibit PSMα production for CA-MRSA. Certain strains of S. aureus are highly sensitive to sub-MICs of protein synthesis inhibitory agents, resulting in an important increase of mRNA levels to overcome the intrinsic ribosome blockage ability of these antibiotics, eventually translating in increased expression of toxins.

The virulome of Streptomyces scabiei in response to cello-oligosaccharide elicitors.

The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the 'green light' to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs' expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei.

Investigate Natural Product Indolmycin and the Synthetically Improved Analogue Toward Antimycobacterial Agents.

Indolmycin (IND) is a microbial natural product that selectively inhibits bacterial tryptophanyl-tRNA synthetase (TrpRS). The tryptophan biosynthesis pathway was recently shown to be an important target for developing new antibacterial agents against Mycobacterium tuberculosis (Mtb). We investigated the antibacterial activity of IND against several mycobacterial model strains. A TrpRS biochemical assay was developed to analyze a library of synthetic IND analogues. The 4″-methylated IND compound, Y-13, showed improved anti-Mtb activity with a minimum inhibitory concentration (MIC) of 1.88 µM (∼0.5 µg/mL). The MIC increased significantly when overexpression of TrpRS was induced in the genetically engineered surrogate M. bovis BCG. The cocrystal structure of Mtb TrpRS complexed with IND and ATP has revealed that the amino acid pocket is in a state between the open form of apo protein and the closed complex with the reaction intermediate. In whole-cell-based experiments, we studied the combination effect of Y-13 paired with different antibacterial agents. We evaluated the killing kinetics, the frequency of resistance to INDs, and the mode of resistance of IND-resistant mycobacteria by genome sequencing. The synergistic interaction of Y-13 with the TrpE allosteric inhibitor, indole propionic acid, suggests that prospective IND analogues could shut down tryptophan biosynthesis and protein biosynthesis in pathogens, leading to a new class of antibiotics. Finally, we discuss a strategy to expand the genome mining of antibiotic-producing microbes specifically for antimycobacterial development.

Putrescine and Its Metabolic Precursor Arginine Promote Biofilm and c-di-GMP Synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.