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Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

Antibiotherapy is the main therapeutic strategy in the fight against bacterial pathogens. However, the misuse of antimicrobials has led to the appearance of antimicrobial-resistant strains. The rate at which we isolate multidrug-resistant bacteria is now much faster than the discovery rate of new antimicrobials. Therefore, the repurposing of approved drugs against multidrug-resistant bacteria is a very promising strategy to find new therapies against these pathogens. Some antibiotics generate oxidative stress as part of their mechanism of action. We have recently applied different methods to find new oxidative stress-producing antimicrobials with synergistic action against intracellular pathogens. Here, we detail several procedures that could be used to identify oxidative stress-producing antimicrobials with a synergistic mechanism of action.

Every year, we face infectious outbreaks produced by harmful microorganisms commonly called superbugs. Often, there is not enough time to find new treatments to cure infected patients. On average, it takes a decade to develop a promising new drug to the point where it can be used on patients! Also, many of the compounds that we identify in the laboratory as promising anti-infectives are not useful for treating patients, mainly because they have unexpected, unsafe side effects. However, researchers have already found thousands of drugs that can safely be used to treat specific diseases. These compounds are approved to be used on patients for particular illnesses, but many of them have not been tested to treat any other diseases. Some of these drugs could be repurposed to treat infections caused by new superbugs. In this article, we summarize some exciting strategies used to find new anti-infectives by drug repurposing.

Staphylococcal infections are a widespread cause of disease in humans. In particular, S. aureus is a major causative agent of infection in clinical medicine. In addition, these bacteria can produce a high number of staphylococcal enterotoxins (SE) that may cause food intoxications. Apart from S. aureus, many coagulase-negative Staphylococcus spp. could be the source of food contamination. Thus, there is an active research work focused on developing novel preventative interventions based on food supplements to reduce the impact of staphylococcal food poisoning. Interestingly, many plant-derived compounds, such as polyphenols, flavonoids, or terpenoids, show significant antimicrobial activity against staphylococci, and therefore these compounds could be crucial to reduce the incidence of food intoxication in humans. Here, we reviewed the most promising strategies developed to prevent staphylococcal food poisoning.

Tuberculosis is the leading cause of death, worldwide, due to a bacterial pathogen. This respiratory disease is caused by the intracellular pathogen Mycobacterium tuberculosis and produces 1.5 million deaths every year. The incidence of tuberculosis has decreased during the last decade, but the emergence of MultiDrug-Resistant (MDR-TB) and Extensively Drug-Resistant (XDR-TB) strains of M. tuberculosis is generating a new health alarm. Therefore, the development of novel therapies based on repurposed drugs against MDR-TB and XDR-TB have recently gathered significant interest. Recent evidence, focused on the role of host molecular factors on M. tuberculosis intracellular survival, allowed the identification of new host-directed therapies. Interestingly, the mechanism of action of many of these therapies is linked to the activation of autophagy (e.g., nitazoxanide or imatinib) and other well-known molecular pathways such as apoptosis (e.g., cisplatin and calycopterin). Here, we review the latest developments on the identification of novel antimicrobials against tuberculosis (including avermectins, eltrombopag, or fluvastatin), new host-targeting therapies (e.g., corticoids, fosfamatinib or carfilzomib) and the host molecular factors required for a mycobacterial infection that could be promising targets for future drug development.

Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. Hijacking specific host molecular factors and/or pathways is necessary for pathogens to successfully establish an intracellular infection. In this study, we employed an unbiased shRNA screening coupled with ultra-fast sequencing to screen 16,000 human genes during S. aureus infection and we identified several host genes important for this intracellular pathogen. In addition, we interrogated our screening results to find novel host-targeted therapeutics against intracellular S. aureus. We found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular bacterial load while host cell viability was restored, showing its importance during intracellular infection. Furthermore, TRAM2 is an interactive partner of the endoplasmic reticulum SERCA pumps and treatment with the SERCA-inhibitor Thapsigargin halted intracellular MRSA survival. Our results suggest that Thapsigargin could be repurposed to tackle S. aureus host cell infection in combination with conventional antibiotics.

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives.

Probiotics have been shown to bind to host receptors, which are important for pathogen adhesion and induce the host’s production of defence factors. They can activate the goblet-cell-derived production of mucins, a major component of the mucus layer and a physical barrier participating in limiting the proximity of microorganisms to the epithelial layer. In the last decade, Bacillus spp. strains have gained interest in human and animal health due to their tolerance and stability under gastrointestinal tract conditions. Moreover, Bacillus spp. strains can also produce various antimicrobial peptides that can support their use as commercial probiotic supplements and functional foods. The present study aimed to evaluate and determine the ability of selected Bacillus spp. strains to inhibit the growth of enterotoxigenic Escherichia coli (ETEC) F4 and to reduce binding of ETEC F4 to HT29-16E (mucus-secreting and goblet-like) human intestinal cells. Moreover, mucus production in the HT29 cells in the presence of the Bacillus spp. strains was quantified by ELISA. Bacillus spp. strains (CHCC 15076, CHCC 15516, CHCC 15541, and CHCC 16872) significantly inhibited the growth of ETEC F4. Moreover, the ability of the probiotic Bacillus spp. strains to stimulate mucin release was highly strain dependent. The treatment with Bacillus subtilis CHCC 15541 resulted in a significant increase of both MUC2 and MUC3 in HT29-16E cells. Therefore, this strain could be an up-and-coming candidate for developing commercial probiotic supplements to prevent infections caused by ETEC F4 and, potentially, other pathogens.

Staphylococcus aureus escapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellular S. aureus are urgently needed. Here, we have studied the physiological changes induced in the host cells by S. aureus during its intracellular proliferation. This is important, because the pathogen exploits the host cell’s metabolism for its own proliferation. We find that S. aureus severely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives., Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection, S. aureus is capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication of S. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism during S. aureus intracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response to S. aureus invasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting that S. aureus induces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition halted S. aureus intracellular proliferation. IMPORTANCE Staphylococcus aureus escapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellular S. aureus are urgently needed. Here, we have studied the physiological changes induced in the host cells by S. aureus during its intracellular proliferation. This is important, because the pathogen exploits the host cell’s metabolism for its own proliferation. We find that S. aureus severely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives.

Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (As

Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (AsV) to arsenite (AsIII) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria.

As a facultative intracellular pathogen, Staphylococcus aureus is able to invade and proliferate within many types of mammalian cells. Intracellular bacterial replication relies on host nutrient supplies and, therefore, cell metabolism is closely bound to intracellular infection. Here, we investigated how S. aureus invasion affects the host membrane-bound fatty acids. We quantified the relative levels of fatty acids and their labelling pattern after intracellular infection by gas chromatography-mass spectrometry (GC-MS). Interestingly, we observed that the levels of three host fatty acids—docosanoic, eicosanoic and palmitic acids—were significantly increased in response to intracellular S. aureus infection. Accordingly, labelling carbon distribution was also affected in infected cells, in comparison to the uninfected control. In addition, treatment of HeLa cells with these three fatty acids showed a cytoprotective role by directly reducing S. aureus growth.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

The coiled-coil protein DivIVA is a determinant of apical growth and hyphal branching in Streptomyces coelicolor. We have investigated the properties of this protein and the involvement of different domains in its essential function and subcellular targeting. In S. coelicolor cell extracts, DivIVA was present as large oligomeric complexes that were not strongly membrane associated. The purified protein could self-assemble into extensive protein filaments in vitro. Two large and conspicuous segments in the amino acid sequence of streptomycete DivIVAs not present in other homologs, an internal PQG-rich segment and a carboxy-terminal extension, are shown to be dispensable for the essential function in S. coelicolor. Instead, the highly conserved amino-terminal of 22 amino acids was required and affected establishment of new DivIVA foci and hyphal branches, and an essential coiled-coil domain affected oligomerization of the protein.

Corynebacterium glutamicum is a rod-shaped actinomycete with a distinct model of peptidoglycan synthesis during cell elongation, which takes place at the cell poles and is sustained by the essential protein DivIVA(CG) (C. glutamicum DivIVA). This protein contains a short conserved N-terminal domain and two coiled-coil regions: CC1 and CC2. Domain deletions and chimeric versions of DivIVA were used to functionally characterize the three domains, and all three were found to be essential for proper DivIVA(CG) function. However, in the presence of the N-terminal domain from DivIVA(CG), either of the two coiled-coil domains of DivIVA(CG) could be replaced by the equivalent coiled-coil domain of Bacillus subtilis DivIVA (DivIVA(BS)) without affecting the function of the original DivIVA(CG), and more than one domain had to be exchanged to lose function. Although no single domain was sufficient for subcellular localization or function, CC1 was mainly implicated in stimulating polar growth and CC2 in targeting to DivIVA(CG) assemblies at the cell poles in C. glutamicum.

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB + plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA + plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA + plasmid but differed over an ≈22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes ( vapB and vapJ to - M , with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA + plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.

Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.

The natural resistance mechanisms of corynebacteria to respond to the environments containing high levels of arsenic were successfully adopted to develop inexpensive and selective extractants for submicrogram amounts of arsenic. Kinetic and equilibrium characteristics were evaluated, and a preliminary exploration of the capability of these strains to be used for arsenic speciation was also made in this work. Three kinetics models were used to fit the experimental data. It was found that the pseudo-first-order kinetics model was not quite adequate to describe the retention process, while the intraparticle diffusion and the pseudo-second-order kinetics models provide the best fits. The equilibrium isotherm showed that the retention of arsenic was consistent with the Langmuir equation and that the Freundlich and Dubinin–Radushkevich models provided poorer fits to the experimental data. The maximum effective retention capacity for arsenic was about 15.4 ng As/mg biomass. The amount of arsenic retained was directly measured in the biomass by forward planning a slurry electrothermal atomic absorption spectrometric procedure.

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.

The genes involved in gluconate catabolism ( gntP and gntK ) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis . In C. glutamicum , gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (P gntK and P gntP ) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or e gfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on P gntP and P gntK fragments was detected only in the presence of cAMP.

Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons ( ars1 and ars2 ) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum . The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein ( arsR ), an arsenite permease ( arsB ), and an arsenate reductase ( arsC ); operon ars1 contains an additional arsenate reductase gene ( arsC1′ ) located immediately downstream from arsC1 . Additional arsenite permease and arsenate reductase genes ( arsB3 and arsC4 ) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1 , arsB1 , arsC1 , arsC1′ , arsR2 , arsB2 , and arsC2 being inducible by arsenite.

The present clinical scenario is one of the growing numbers of immunocompromised patients infected with a variety of fungal pathogens. AIDS, tuberculosis, immunosuppressive therapy, cancer chemotherapy, or the use of broad-spectrum antibiotics contribute to the boost of such patient category. However, progress in the treatment of fungal infections has been slow. Different polyenes and/or azoles are available for the treatment of systemic fungal infections, but it is becoming clear that new and more effective antifungal agents are urgently required. Fortunately, biosynthetic gene clusters for several antifungal polyene macrolides have been characterized, and this opened the way to generate improved antifungal compounds via genetic engineering as well as to understand the molecular mechanisms that regulate polyene production. Here, we will review the history of the discovery of antifungal polyenes, their environmental role, and the strategies currently used for their identification and improvement.

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection., This work was funded by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (Refs SAF2008‐00350 and SAF2011‐23714), Fundación Solórzano, Junta de Castilla y León (Consejería de Educación, Ref. CSI001A10‐2, and Consejería de Sanidad) and Consejo Superior de Investigaciones Científicas (CSIC; Ref. 200720I026). Additional funding comes from the FEDER programme of the European Union. EB is a graduate student funded by a predoctoral fellowship from the FPU programme (Ministerio de Educación, MEC, Spanish Government). ML is funded by JAE‐Doc and Juan de la Cierva postdoctoral contracts (MEC and Social European Fund of the European Union, 2007–2013). AF is funded by a long‐term EMBO postdoctoral fellowship and a Juan de la Cierva contract. KP is a graduate student funded by an FPI fellowship (MEC).

Bacterial cell size and morphology are enormously diverse. The molecular factors of morphogenesis are well understood in certain bacterial models and fairly conserved throughout a broad spectrum of bacterial species, as follows. In most bacteria, the tubulin-like FtsZ protein polymerizes at the mid cell , thereby generating the scaffold of the bacterial cell division septum. Actin-like MreB homologues are required for cell elongation at the lateral walls of Escherichia coli or Bacillus subtilis. Whereas FtsZ is conserved in Corynebacterium glutamicum, mreB homologues are absent in the corynebacterial genomes sequenced to date. Furthermore, in these bacteria, cell elongation occurs at the polar ends in a mycelial fashion. This process is structurally maintained from the inside of the cell by oligomers created through self-interaction of DivIVA, a coiled-coil-rich cytoskeletal protein that interacts with the molecular machinery of the polar cell wall synthesis. In addition, the molecular factors involved in the spatio-temporal regulation of bacterial cell division are also missing in C. glutamicum. However, certain serine/threonine kinases have been reported recently in this organism that could be implicated in a tight regulation of cytokinesis through protein phosphorylation. Since numerous antibiotics target bacterial cell division or cell elongation genes, a detailed understanding of these processes could enable the development of novel antibiotics for treating bacterial infections caused by pathogenic Corynebacteria or by the closely related Mycobacteria, Nocardia, or Rhodococcus.

Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known aboutActinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such asMycobacterium, Corynebacterium, andStreptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals.

Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.

Rhodococcus equi is the only animal pathogen among the rhodococci. A soil inhabitant, R. equi is prevalent in the farm environment where it uses herbivore manure as growth substrate. In addition to its saprophytic lifestyle, R. equi has the ability to colonize animal host tissues, causing pyogranulomatous infections in a variety of mammals. Although clearly a multihost pathogen, R. equi is best known as the etiologic agent of a severe contagious bronchopneumonic disease in horses. It also causes deadly opportunistic infections in immunosuppressed individuals, particularly tuberculosis-like cavitary pneumonia in HIV-infected patients. R. equi is an intracellular parasite that replicates within a modified phagocytic vacuole in macrophages. Its pathogenicity depends on a horizontally-acquired genomic island carried on a virulence plasmid. This plasmid virulence locus encodes a family of surface-associated antigens, the Vap proteins. The products of the vap island confer the ability to survive within macrophages and recent evidence suggests they are also involved in infectious tropism towards specific animal host species (horses, pigs, and cattle). The complete DNA sequence of the R. equi genome has been recently determined and a detailed analysis will soon be published. This chapter reviews the current, “pregenomic” state of knowledge about the biology and virulence of this fascinating pathogenic actinomycete.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi., Author Summary Rhodococcus is a prototypic genus within the Actinobacteria, one of the largest microbial groups on Earth. Many of the ubiquitous rhodococcal species are biotechnologically useful due to their metabolic versatility and biodegradative properties. We have deciphered the genome of a facultatively parasitic Rhodococcus, the animal and human pathogen R. equi. Comparative genomic analyses of related species provide a unique opportunity to increase our understanding of niche-adaptive genome evolution and specialization. The environmental rhodococci have much larger genomes, richer in metabolic and degradative pathways, due to gene duplication and acquisition, not genome contraction in R. equi. This probably reflects that the host-associated R. equi habitat is more stable and favorable than the chemically diverse but nutrient-poor environmental niches of nonpathogenic rhodococci, necessitating metabolically more complex, expanded genomes. Our work also highlights that the recruitment or cooption of core microbial traits, following the horizontal acquistion of a few critical genes that provide access to the host niche, is an important mechanism in actinobacterial virulence evolution. Gene cooption is a key evolutionary mechanism allowing rapid adaptive change and novel trait acquisition. Recognizing the contribution of cooption to virulence provides a rational framework for understanding and interpreting the emergence and evolution of microbial pathogenicity.

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces .