RESUMEN
Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.
Asunto(s)
Acenaftenos , Topoisomerasa de ADN IV , Escherichia coli , Compuestos Heterocíclicos con 3 Anillos , Aminoácidos/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/genéticaRESUMEN
Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wild-type gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water-metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wild-type and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.
Asunto(s)
Ciprofloxacina , Gonorrea , Humanos , Ciprofloxacina/farmacología , Fluoroquinolonas/farmacología , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , Neisseria gonorrhoeae , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Girasa de ADN/genética , Girasa de ADN/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV but interact differently with the enzymes. This has led to the development of the "novel bacterial topoisomerase inhibitor" (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases, including gyrase from Mycobacterium tuberculosis and gyrase and topoisomerase IV from Bacillus anthracis and Escherichia coli. GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.
Asunto(s)
Antibacterianos/química , División del ADN/efectos de los fármacos , Indoles/química , Naftiridinas/química , Piperidinas/química , Piridonas/química , Inhibidores de Topoisomerasa II/química , Bacillus anthracis/enzimología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Girasa de ADN/química , Topoisomerasa de ADN IV/antagonistas & inhibidores , ADN Bacteriano/efectos de los fármacos , ADN de Cadena Simple/efectos de los fármacos , Escherichia coli/enzimología , Mycobacterium tuberculosis/enzimologíaRESUMEN
A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Girasa de ADN/metabolismo , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Línea Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/químicaRESUMEN
Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 µM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 µM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.
Asunto(s)
Acenaftenos/química , Acenaftenos/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Girasa de ADN/química , Girasa de ADN/genética , Girasa de ADN/metabolismo , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
This corrects the article DOI: 10.1038/ncomms16081.
RESUMEN
The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Descubrimiento de Drogas/métodos , Biblioteca de Genes , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Staphylococcus aureus/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Evaluación Preclínica de Medicamentos , Terapia Molecular Dirigida , Mycobacterium tuberculosis/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.
Asunto(s)
Antibacterianos , División del ADN , Girasa de ADN , Tiofenos , Antibacterianos/química , Antibacterianos/metabolismo , Cristalografía por Rayos X , Girasa de ADN/química , Girasa de ADN/metabolismo , Descubrimiento de Drogas , Modelos Moleculares , Tiofenos/química , Tiofenos/metabolismoRESUMEN
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II/química , Antibacterianos/farmacología , Antineoplásicos/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana , Etopósido/farmacología , Fluoroquinolonas/farmacología , Modelos Moleculares , Estructura Molecular , Moxifloxacino , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15â Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.
Asunto(s)
División del ADN , Girasa de ADN/química , Etopósido/química , Fluoroquinolonas/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos de Espiro/química , Staphylococcus aureus/enzimología , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , MoxifloxacinoRESUMEN
DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
Asunto(s)
Antígenos de Neoplasias/aislamiento & purificación , Clonación Molecular/métodos , ADN-Topoisomerasas de Tipo II/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Adenosina Difosfato/metabolismo , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Baculoviridae/genética , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , ADN Circular/química , ADN Circular/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Concentración 50 Inhibidora , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Espectrometría de Fluorescencia , Spodoptera/metabolismoRESUMEN
Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.
Asunto(s)
Acinetobacter baumannii/enzimología , Topoisomerasa de ADN IV/química , Inhibidores Enzimáticos/química , Quinolonas/química , Topoisomerasa de ADN IV/farmacología , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Quinolonas/farmacologíaRESUMEN
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Girasa de ADN/química , Quinolinas/química , Quinolinas/farmacología , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II , Antibacterianos/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , División del ADN , Girasa de ADN/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Diseño de Fármacos , Resistencia a Medicamentos , Escherichia coli/enzimología , Manganeso/metabolismo , Modelos Moleculares , Conformación Proteica , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Relación Estructura-ActividadRESUMEN
The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.
Asunto(s)
Amidohidrolasas , Antibacterianos/farmacología , Fucosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Esenciales , Regiones Promotoras Genéticas/genética , Streptococcus pneumoniae/efectos de los fármacos , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Inhibidores Enzimáticos/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Datos de Secuencia Molecular , Operón , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/metabolismoRESUMEN
The appearance of antibiotic resistant pathogens, including vancomycin resistant Staphylococcus aureus, in the clinic has necessitated the development of new antibiotics. The golden age of antibiotic discovery, in which potent selective compounds were readily extracted from natural product extracts is over and novel approaches need to be implemented to cover the therapeutic shortfall. The generation of huge quantities of bacterial sequence data has allowed the identification of all the possible targets for therapeutic intervention and allowed the development of screens to identify inhibitors. Here, we described a number of target classes in which genomics has contributed to its identification. As a result of analyzing sequence data, all of the tRNA synthetases and all of the two-component signal transduction systems were readily isolated; which would not have been easily identified if whole genome sequences were not available. Fatty acid biosynthesis is a known antibacterial target, but genomics showed which genes in that pathway had the appropriate spectrum to be considered as therapeutic targets. Genes of unknown function may seem untractable targets, but if those that are broad spectrum and essential are identified, it becomes valuable to invest time and effort to determine their cellular role. In addition, we discuss the role of genomics in developing technologies that assist in the discovery of new antibiotics including microarray gridding technology. Genomics can also increase the chemical diversity against which the novel targets can be screened.
Asunto(s)
Antibacterianos/química , Diseño de Fármacos , Genoma Bacteriano , Genómica , Técnicas Químicas Combinatorias , Genómica/tendencias , HumanosRESUMEN
Staphylococcus aureus is a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulence-determinant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator (sarA), alpha-haemolysin (hla), surface protein A (spa) and toxic-shock syndrome toxin-1 (tst) genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression of hla, tst and spa was strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), but sarA was relatively unaffected. The global regulator of expression of virulence-determinant genes, agr (accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression of tst in wild-type or agr backgrounds and failed to relieve the salt suppression. Expression of tst was strongly stimulated in several low-metal environments, independently of agr, whilst spa levels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.