Ferric Uptake Regulator Fur Coordinates Siderophore Production and Defense against Iron Toxicity and Oxidative Stress and Contributes to Virulence in Chromobacterium violaceum.

Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the ferric uptake regulator Fur in the physiology and virulence of C. violaceum A Δfur mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR-Cas locus rescued the Δfur mutant growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceumIMPORTANCE Maintenance of iron homeostasis, i.e., avoiding both deficiency and toxicity of this metal, is vital to bacteria and their hosts. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the ferric uptake regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum We determined that fur is a conditionally essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation, and siderophore production. Consistent with these results, a C. violaceumfur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.

A Chemical Counterpunch: Chromobacterium violaceum ATCC 31532 Produces Violacein in Response to Translation-Inhibiting Antibiotics.

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.

Production and Uptake of Distinct Endogenous Catecholate-Type Siderophores Are Required for Iron Acquisition and Virulence in Chromobacterium violaceum.

Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of Chromobacterium violaceum, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that C. violaceum produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An in silico analysis in the genome of C. violaceum revealed that genes related to synthesis and uptake of chromobactin (cba) and viobactin (vba) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of C. violaceum, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence in vivo and reduced neutrophil extracellular trap (NET) formation in in vitro assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that C. violaceum uses distinct endogenous siderophores for iron uptake and its establishment in the host.

Chromobacterium violaceum and Pseudomonas aeruginosa PAO1: Models for Evaluating Anti-Quorum Sensing Activity of Melaleuca alternifolia Essential Oil and Its Main Component Terpinen-4-ol.

The problem of antibiotic resistance among pathogens encourages searching for novel active molecules. The aim of the research was to assay the anti-quorum sensing (anti-QS) and antibiofilm potential of Melaleuca alternifolia essential oil and its main constituent, terpinen-4-ol, to prevent the infections due to methicillin-resistant Staphylococcus aureus strains as an alternate to antibiotics. The tea tree oil (TTO) was evaluated for its potential in inhibiting QS-dependent phenomena such as violacein production in Chromobacterium violaceum, swarming motility of Pseudomonas aeruginosa PAO1, and biofilm formation in MRSA strains on glass. The results showed that terpinen-4-ol was able to inhibit MRSA strain biofilm formation on the glass strips by 73.70%. TTO inhibited the violacein production at a mean inhibitory concentration (MIC) value of 0.048 mg/mL by 69.3%. At 100 µg/mL TTO and terpinen-4-ol exhibited inhibition in swarming motility of PAO1 by 33.33% and 25%, respectively. TTO revealed anti-QS and anti-biofilm activities at very low concentrations, but it could be further investigated for new molecules useful for the treatment of MRSA infections.

Identification of Chromobacterium violaceum by MALDI-TOF mass spectrometry in blood culture

Report of Chromobacterium violaceum isolation from blood culture. Identification by MALDI-TOF mass spectrometry. Relevant report due to the site affected, infection severity, and importance of correct and rapid identification for a successful treatment and lower risk of morbidity and mortality. (AU)

Action and function of Chromobacterium violaceum in health and disease: Violacein as a promising metabolite to counteract gastroenterological diseases.

Chromobacterium violaceum is a Gram negative, ß-proteobacterium found in the microbiota of tropical and subtropical environments. Although considered an opportunistic pathogen, infection rapidly progress to fatal sepsis, with metastatic abscesses. It is noteworthy the multidrug resistant phenotype of C. violaceum and the possibility of relapse. Recently, an influence of global climate in the incidence of cases beyond the previous areas has been observed. Furthermore, chronic granulomatous disease has been considered a risk factor to infection. Despite the increase in C. violaceum infection incidence and high mortality, most clinicians are not familiar with it. This review pointed out important features of this life threatening microorganism, including its pathogenicity, mechanistic aspects, genetic and drug resistance associated factors, and the clinical association with chronic granulomatous disease. In addition, its main metabolite violacein may be a promising agent to counteract gastroenterological diseases, such as colorectal cancer and inflammatory gastric lesions.

Genetic variability in isolates of Chromobacterium violaceum from pulmonary secretion, water, and soil.

Chromobacterium violaceum is a free-living Gram-negative bacillus usually found in the water and soil in tropical regions, which causes infections in humans. Chromobacteriosis is characterized by rapid dissemination and high mortality. The aim of this study was to detect the genetic variability among C. violaceum type strain ATCC 12472, and seven isolates from the environment and one from a pulmonary secretion from a chromobacteriosis patient from Ilhéus, Bahia. The molecular characterization of all samples was performed by polymerase chain reaction (PCR) sequencing and 16S rDNA analysis. Primers specific for two ATCC 12472 pathogenicity genes, hilA and yscD, as well as random amplified polymorphic DNA (RAPD), were used for PCR amplification and comparative sequencing of the products. For a more specific approach, the PCR products of 16S rDNA were digested with restriction enzymes. Seven of the samples, including type-strain ATCC 12472, were amplified by the hilA primers; these were subsequently sequenced. Gene yscD was amplified only in type-strain ATCC 12472. MspI and AluI digestion revealed 16S rDNA polymorphisms. This data allowed the generation of a dendogram for each analysis. The isolates of C. violaceum have variability in random genomic regions demonstrated by RAPD. Also, these isolates have variability in pathogenicity genes, as demonstrated by sequencing and restriction enzyme digestion.

Tea polyphenols as an antivirulence compound Disrupt Quorum-Sensing Regulated Pathogenicity of Pseudomonas aeruginosa.

Green tea, a water extract of non-fermented leaves of Camellia sinensis L., is one of the nonalcoholic beverages in China. It is becoming increasingly popular worldwide, because of its refreshing, mild stimulant and medicinal properties. Here we examined the quorum sensing inhibitory potentials of tea polyphenols (TP) as antivirulence compounds both in vitro and in vivo. Biosensor assay data suggested minimum inhibitory concentrations (MICs) of TP against selected pathogens were 6.25 ~ 12.5 mg/mL. At sub-MIC, TP can specifically inhibit the production of violacein in Chromobacterium violaceum 12472 with almost 98% reduction at 3.125 mg/mL without affecting its growth rate. Moreover, TP exhibited inhibitory effects on virulence phenotypes regulated by QS in Pseudomonas aeruginosa. The total proteolytic activity, elastase, swarming motility and biofilm formation were reduced in a concentration-dependent manner. In vivo, TP treatment resulted in the reduction of P. aeruginosa pathogenicity in Caenorhabditis elegans. When its concentration was 3.125 mg/mL, the survival rate reached 63.3%. In the excision wound infection model, the wound contraction percentage in treatment groups was relatively increased and the colony-forming units (CFU) in the wound area were significantly decreased. These results suggested that TP could be developed as a novel non-antibiotic QS inhibitor without killing the bacteria but as an antivirulence compound to control bacterial infection.

Chromobacterium violaceum: um caso fatal no nordeste do Brasil / Chromobacterium violaceum: a fatal case in the northeast of the Brazil

Chromobacterium violaceum is a rare pathogen that can potentially cause fatal infections in humans. An 8-year-old child from Natal, northeast of Brazil, presented history of fever, sore throat, and abdominal pain, during 5 days before admission, and died 4 hours after hospitalization. Chromobacterium violaceum was isolated from oropharynx scrapings and was resistant to ampicillin, cefotaxime, cefalotin, ceftazidime, and ceftriaxone...

Chromobacterium violaceum é um patógeno raro que potencialmente pode causar infecções fatais em humanos. Relatamos o caso de uma criança de 8 anos de idade, moradora da cidade de Natal, nordeste do Brasil, que apresentou história de febre, dor na garganta e no abdome durante os cinco dias anteriores à internação, e veio a falecer após 4 horas de hospitalização. A bactéria Chromobacterium violaceum foi isolada da orofaringe e apresentou resistência a ampicilina, cefotaxima, cefalotina, ceftazidima e ceftriaxona...

The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen.

Computational analysis suggests that virulence of Chromobacterium violaceum might be linked to biofilm formation and poly-NAG biosynthesis

Groups of genes that produce exopolysaccharide with a N-acetyl-D-glucosamine monomer are in the genome of several pathogenic bacteria. Chromobacterium violaceum, an opportunistic pathogen, has the operon hmsHFR-CV2940, whose proteins can synthesize such polysaccharide. In this work, multiple alignments among proteins from bacteria that synthesize such polysaccharide were used to verify the existence of amino acids that might be critical for pathogen activity. Three-dimensional models were generated for spatial visualization of these amino acid residues. The analysis carried out showed that the protein HmsR preserves the amino acids D135, D228, Q264 and R267, considered critical for the formation of biofilms and, furthermore, that these amino acids are close to each other. The protein HmsF of C. violaceum preserves the residues D86, D87, H156 and W115. It was also shown that these residues are also close to each other in their spatial arrangement. For the proteins HmsH and CV2940 there is evidence of conservation of the residues R104 and W94, respectively. Conservation and favorable spatial location of those critical amino acids that constitute the proteins of the operon indicates that they preserve the same enzymatic function in biofilm synthesis. This is an indicator that the operon hmsHFR-CV2940 is a possible target in C. violaceum pathogenicity.

Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity

Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil. Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals. Although rare, infections with C. violaceum are characterized by rapid dissemination and high mortality. With the complete genome sequence of C. violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed. Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged. The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis. We described a number of genes involved in infectious processes, such as host cell adhesion, [quot ]contact-dependent secretion[quot ] of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins. We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response

Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria.

Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere. Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles. Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus: all these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics. One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes. In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens. In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers. These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.

Phagocyte NADPH oxidase, but not inducible nitric oxide synthase, is essential for early control of Burkholderia cepacia and chromobacterium violaceum infection in mice.

Reactive oxygen and nitrogen intermediates have critical, partially overlapping roles in host defense against a variety of pathogens. Using mice deficient in generating phagocyte superoxide (p47(phox)(-/-)) and mice deficient in generating inducible nitric oxide synthase (iNOS(-/-)), we examined the roles of these reactive species in host defense against Burkholderia cepacia and Chromobacterium violaceum, organisms known to have unusual virulence in chronic granulomatous disease. Intraperitoneal B. cepacia challenge (4.0 x 10(3) to 4.0 x 10(5) organisms/mouse) resulted in mortality in all p47(phox)(-/-) mice, with the survival interval being inversely proportionate to the amount of inoculum. Pretreatment with gamma interferon did not affect survival. C. violaceum was strikingly virulent in p47(phox)(-/-) mice (the 50% lethal dose [LD(50)] was <13 organisms). iNOS(-/-) and wild-type mice were resistant to B. cepacia challenges of at least 10(6) organisms per mouse, and the LD(50) of C. violaceum was between 10(6) and 10(7) organisms per mouse. Consistent with the survival data, numbers of organisms in cultures of B. cepacia from multiple sites were higher for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice at day 4 after challenge, but numbers of organisms for different B. cepacia strains varied. The recovery of C. violaceum was strikingly greater at 18 h after challenge for p47(phox)(-/-) mice than for iNOS(-/-) and wild-type mice, in which the organism burdens were virtually nil. In vitro, both B. cepacia and C. violaceum were sensitive to H(2)O(2) and to reactive nitrogen intermediates but the sensitivities of different strains varied significantly. Host defense against B. cepacia and C. violaceum is critically dependent in vivo on reactive oxygen intermediates, and these species are model organisms to further dissect host and pathogen interactions related to the generation and scavenging of microbicidal reactive intermediates.