Stress response and virulence factors in bacterial pathogens relevant for Chilean aquaculture: current status and outlook of our knowledge

The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.

CdsH Contributes to the Replication of Salmonella Typhimurium inside Epithelial Cells in a Cysteine-Supplemented Medium.

Salmonella Typhimurium is a facultative, intracellular pathogen whose products range from self-limited gastroenteritis to systemic diseases. Food ingestion increases biomolecules' concentration in the intestinal lumen, including amino acids such as cysteine, which is toxic in a concentration-dependent manner. When cysteine's intracellular concentration reaches toxic levels, S. Typhimurium expresses a cysteine-inducible enzyme (CdsH), which converts cysteine into pyruvate, sulfide, and ammonia. Despite this evidence, the biological context of cdsH's role is not completely clear, especially in the infective cycle. Since inside epithelial cells both cdsH and its positive regulator, ybaO, are overexpressed, we hypothesized a possible role of cdsH in the intestinal phase of the infection. To test this hypothesis, we used an in vitro model of HT-29 cell infection, adding extra cysteine to the culture medium during the infective process. We observed that, at 6 h post-invasion, the wild type S. Typhimurium proliferated 30% more than the ΔcdsH strain in the presence of extra cysteine. This result shows that cdsH contributes to the bacterial replication in the intracellular environment in increased concentrations of extracellular cysteine, strongly suggesting that cdsH participates by increasing the bacterial fitness in the intestinal phase of the S. Typhimurium infection.

The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human ß-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.

CysB-dependent upregulation of the Salmonella Typhimurium cysJIH operon in response to antimicrobial compounds that induce oxidative stress.

It has been proposed that some antibiotics exert additional damage through reactive oxygen species (ROS) production. Since H2S protects neurons and cardiac muscle from oxidative stress, it has been hypothesized that bacterial H2S might, similarly, be a cellular protector against antibiotics. In Enterobacteriaceae, H2S can be produced by the cysJIH pathway, which uses sulfate as the sulfur source. CysB, in turn, is a positive regulator of cysJIH. At present, the role of S. Typhimurium cysJIH operon in the protection to reactive oxygen species (ROS) induced by antimicrobial compounds remains to be elucidated. In this work, we evaluated the role of cysJIH and cysB in ROS accumulation, superoxide dismutase (SOD) activity, reduced thiol accumulation, and H2S accumulation in S. Typhimurium, cultured in either sulfate or cysteine as the sole sulfur source. Furthermore, we assessed the effects of the addition of ceftriaxone (CEF) and menadione (MEN) in these same parameters. In sulfate as the sole sulfur source, we found that the cysJIH operon and the cysB gene were required to full growth in minimal media, independently on the addition of CEF or MEN. Most importantly, both cysJIH and cysB contributed to diminish ROS levels, increase the SOD activity, increase the reduced thiols, and increase the H2S levels in presence of CEF or MEN. Moreover, the cysJIH operon exhibited a CysB-dependent upregulation in presence of these two antimicrobials compounds. On the other hand, when cysteine was used as the sole sulfur source, we found that cysJIH operon was completely negligible, were only cysB exhibited similar phenotypes than the described for sulfate as sulfur source. Unexpectedly, CysB downregulated cysJIH operon when cysteine was used instead of sulfate, suggesting a complex regulation of this system.

Microarray analysis of the Escherichia coli response to CdTe-GSH Quantum Dots: understanding the bacterial toxicity of semiconductor nanoparticles.

BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are made of heavy metals and involve synthetic methods that require organic solvents and high temperatures. This issue makes the development of water-soluble nanoparticles with lower toxicity a major topic of interest. In a previous work our group described a biomimetic method for the aqueous synthesis of CdTe-GSH Quantum Dots (QDs) using biomolecules present in cells as reducing and stabilizing agents. This protocol produces nanoparticles with good fluorescent properties and less toxicity than those synthesized by regular chemical methods. Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so it is important to know in detail the effects of these semiconductor nanoparticles on cells, their levels of toxicity and the strategies that cells develop to overcome it. RESULTS: In this work, the response of E. coli exposed to different sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through transcriptomic, biochemical, microbiological and genetic approaches. It was determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii) QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA) and tellurium does not contribute significantly to QDs-mediated toxicity since cells incorporate low levels of Te, iii) red QDs also induce genes related to oxidative stress response and membrane proteins, iv) Cd2+ release is higher in red QDs, and v) QDs render the cells more sensitive to polymyxin B. CONCLUSION: Based on the results obtained in this work, a general model of CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial toxicity of QDs is mainly associated with cadmium release, oxidative stress and loss of membrane integrity. The higher toxicity of red QDs is most probably due to higher cadmium content and release from the nanoparticle as compared to green QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making these biomimetic QDs candidates for adjuvant therapies against bacterial infections.

Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

Cloning, purification and characterization of Geobacillus stearothermophilus V uroporphyrinogen-III C-methyltransferase: evaluation of its role in resistance to potassium tellurite in Escherichia coli.

The Geobacillus stearothermophilus V cobA gene encoding uroporphyrinogen-III C-methyltransferase (also referred to as SUMT) was cloned into Escherichia coli and the recombinant enzyme was overexpressed and purified to homogeneity. The enzyme binds S-adenosyl-L-methionine and catalyzes the production of III methyl uroporphyrinogen in vitro. E. coli cells expressing the G. stearothermophilus V cobA gene exhibited increased resistance to potassium tellurite and potassium tellurate. Site-directed mutagenesis of cobA abolished tellurite resistance of the mesophilic, heterologous host and SUMT activity in vitro. No methylated, volatile derivatives of tellurium were found in the headspace of tellurite-exposed cobA-expressing E. coli, suggesting that the role of SUMT methyltransferase in tellurite(ate) detoxification is not related to tellurium volatilization.

Catalases are NAD(P)H-dependent tellurite reductases.

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

The Geobacillus stearothermophilus V iscS gene, encoding cysteine desulfurase, confers resistance to potassium tellurite in Escherichia coli K-12.

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters.