Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.

Novel anti-tubercular and antibacterial based benzosuberone-thiazole moieties: Synthesis, molecular docking analysis, DNA gyrase supercoiling and ATPase activity.

Herein, molecular hybridization strategy was utilized in the design of new benzosuberone-thiazole derivatives. The structures of the synthesized hybrids were determined on the basis of elemental and spectral analyses. These compounds were evaluated for their antibacterial activities against five bronchitis causing bacteria in addition to their anti-tubercular activities. Most compounds revealed promising activities. Amongst active compounds, benzosuberone-dithiazole derivatives 22a and 28 with MIC value = 1.95 µg/ml against H. influenza, M. pneumonia, and B. pertussis displayed four times the activity of ciprofloxacin (MIC = 7.81 µg/ml) against H. influenza, twice the activity of ciprofloxacin (MIC = 3.9 µg/ml) against M. pneumonia and were equipotent to ciprofloxacin against B. pertussis (MIC = 1.95 µg/ml). Additionally, benzosuberone-dithiazole derivatives 22a and 27 were the most promising anti-tubercular among the tested compounds with MIC values of 0.12 and 0.24 µg/ml, respectively against sensitive M. tuberculosis in addition to high activity against resistant strain of M. tuberculosis (MIC = 0.98 and 1.95 µg/ml, respectively) compared to isoniazid (MIC = 0.12 µg/ml against sensitive M. tuberculosis and no activity against resistant M. tuberculosis). Cytotoxicity study of the active dithiazole derivatives 22a, 27 and 28 against normal human lung cells (WI-38) indicated their high safety profile as showed from their high IC50 values (IC50 = 107, 74.8, and 117 µM, respectively). Furthermore, DNA gyrase supercoiling and ATPase activity assays showed that 22a, 27 and 28 have the potential to inhibit DNA gyrase at low micromolar levels (IC50 = 3.29-15.64 µM). Molecular docking analysis was also carried out to understand the binding profiles of the synthesized compounds into the ATPase binding sites of bacterial and mycobacterial DNA gyraseB.

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin.

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

A Bordetella pertussis MgtC homolog plays a role in the intracellular survival.

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis.

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

BACKGROUND INFORMATION: The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. RESULTS: In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 µm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). CONCLUSIONS: MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. SIGNIFICANCE: These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis.

Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production.

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 µg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002

Whooping cough or pertussis was a major cause of childhood morbidity and mortality in the world until the introduction of a whole-cell vaccine in the 1940's. However, since the early 1980's whooping cough cases have increased in many countries, becoming an important problem of public health. This increase may be due to accuracy of laboratory diagnosis and reporting of the disease, a decline in immunity over time, demographic changes, and adaptation of the bacterial population to vaccine-induced immunity. The purpose of this study was to analyze phenotypically and genotypically a collection of 67 Bordetella pertussis isolates recovered during the period 1988-2002 in São Paulo State, Brazil to determine their characteristics and relatedness. All isolates were submitted to susceptibility testing to erythromycin, serotyping, and 56 isolates were analyzed by Pulsed Field Gel Electrophoresis (PFGE). All isolates were susceptible to erythromycin and the majority of them belonged to serotype 1,3. The 56 isolates were classified into 11 PFGE profiles according to the differences in banding patterns. Although more than 60 percent of the isolates were recovered from patients aged less than three months, almost 15 percent of them were isolated from adolescents/adults evidencing the increase in the incidence of pertussis among this group of age.

A coqueluche ou pertussis foi a maior causa de morbidade e mortalidade infantil em todo o mundo até a introdução de uma vacina na década de 1940. Entretanto, desde a década de 1980, a coqueluche tornou-se, em muitos países , um importante problema de saúde pública. Este acontecimento pode ser atribuído à melhoria do diagnóstico laboratorial e da notificação da doença, declínio da imunidade no decorrer do tempo, mudanças demográficas ou adaptação da população bacteriana à imunidade induzida pela vacina. O objetivo deste estudo foi analisar as características fenotípicas e genotípicas de uma coleção de 67 cepas de Bordetella pertussis isoladas no período 1988-2002 em São Paulo, Brasil. Todas as cepas foram submetidas à determinação do perfil de resistência à eritromicina, à sorotipagem e 56 cepas à eletroforese em campo pulsado (PFGE). Todas as cepas foram sensíveis à eritromicina e a maioria delas pertencia ao sorotipo 1,3. As 56 cepas foram classificadas em 11 perfis de PFGE com base nas diferenças no padrão de bandas. Embora mais de 60 por cento das cepas tenham sido isoladas de crianças com menos de três meses de idade, cerca de 15 por cento delas era de adolescentes/adultos evidenciando um aumento da incidência da coqueluche nesse grupo etário.

Expression of the lipopolysaccharide-modifying enzymes PagP and PagL modulates the endotoxic activity of Bordetella pertussis.

Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.

Effect of relevant culture parameters on Pertussis Toxin expression by Bordetella pertussis.

Whooping cough vaccines are produced using different ranges of cultivation conditions and medium compositions, which are known to influence growth rate, virulence factor production and degradation, as well as the virulence factors' association to the cell. This study quantifies the impact of individual parameters on Pertussis Toxin (PT) production, using an optimized chemically defined medium as starting point, rather than a complex medium. A number of chemicals that are identified affect both growth rate and virulence factor production, which occur at similar levels in various commonly used production media. Also, degradation by proteolytic activity is shown to be an important parameter to monitor, since it significantly affects the PT yield. Low sodium concentrations, i.e. 50-75 mM rather than the conventional 100-140 mM, significantly increase the growth rate of the organism, the final optical density, as well as the association of PT to the cells. The absolute amount of biomass produced measured as dry weight, is similar for all sodium concentrations tested, contrary to earlier work. While it is known that high iron concentrations inhibit virulence factor production, it is shown here that iron-limited growth results in very high specific PT production. This finding may be used to produce a whole-cell vaccine with little biomass per dose, reducing whole-cell vaccine toxicity. The Bordetella pertussis strain 509 used here produces 30% more PT at 34 than at 37 degrees C, a commonly used cultivation temperature. The data in this study show that existing production processes for cellular and acellular vaccines can in principle be optimised considerably by taking simple measures.

Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis.

Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.

Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-gamma production in vivo.

We have previously demonstrated that Fasciola hepatica infection significantly reduced Bordetella pertussis-specific interferon (IFN)-gamma production in mice coinfected with B. pertussis or immunized with a pertussis whole cell vaccine (Pw). In the present study, we have identified parasite molecules capable of mimicking this suppressive effect of F. hepatica. Parenteral injection of mice with culture medium in which adult F. hepatica were maintained (excretory/secretory, ES, products) suppressed B. pertussis-specific IFN-gamma production in mice immunized with Pw. The suppressive effect of ES was abrogated by coinjecting ES with the cysteine proteinase inhibitor, Z-Phe-Ala-diazomethylketone. Furthermore, purified cathepsin L proteinase (FheCL), a major component of ES products, was capable of suppressing IFN-gamma production. The suppressive effect of FheCL was attenuated in interleukin (IL)-4 defective (IL-4-/-) mice. Therefore, FheCL released by F. hepatica is involved in the suppression of Th1 immune responses and this suppression may be dependent upon IL-4.

En coqueluche: ¿es posible? / In pertussis: is it possible?

Existen esquemas alternativos a los recomendados por la Academia Americana de Pediatría para el tratamiento antimicrobiano de la coqueluche. Estudios controlados apoyan la posibilidad de reducir el plazo de tratamiento a 7 días empleando eritromicina. Desde cuidarse en especial la dosficación de eritromicina (para etilsuccinato debe ser de 50 a 60 mg/kg/ día y administrarse siempre inmediatamente después de las comidas). El empleo de nuevos macrólidos requiere mayor base experimental para establecer dosis diaria, duración y eficacia bacteriológica. Es conveniente implementar el cultivo en centros centros centinelas para vigilar la susceptibilidad in vitro de bordetella pertussis y ratificar la eficacia bacteriológica de los esquemas abreviados de terapia específica. Los pasos abreviados de tratamiento de la infección establecida por bordetella pertussis parecen ser extrapolables a los esquemas recomendados para la profilaxis en los contactos

Disruption of tonB in Bordetella bronchiseptica and Bordetella pertussis prevents utilization of ferric siderophores, haemin and haemoglobin as iron sources.

The Bordetella bronchiseptica tonB gene was cloned by detection of a chromosomal restriction fragment hybridizing with each of two degenerate oligonucleotides that corresponded to Pro-Glu and Pro-Lys repeats characteristic of known TonB proteins. The tonB(Bb) gene was situated upstream of exbB and exbD homologues and downstream of a putative Fur-regulated promoter. Hybridization results indicated that the tonB operon and flanking regions were highly conserved between B. bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Disruption of tonB in B. bronchiseptica resulted in inability to grow in iron-limiting media, and inability to utilize alcaligin, enterobactin, ferrichrome, desferroxamine B, haemin and haemoglobin. Although it was not possible to inactivate tonB in a clinical B. pertussis isolate, tonB was disrupted in a laboratory B. pertussis strain previously selected for the ability to grow on Luria-Bertani medium. This B. pertussis tonB mutant shared a similar iron complex utilization deficient phenotype with the B. bronchiseptica tonB mutant. The B. bronchiseptica tonB operon present on a plasmid did not complement an Escherichia coli tonB mutant, but inefficient reconstitution of enterobactin utilization was observed in one fepA mutant harbouring plasmid copies of the B. pertussis fepA homologue and tonB(Bb) operon.

The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides.

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.

Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes.

Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-Smal Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.

Susceptibilities of Bordetella pertussis strains to antimicrobial peptides.

We examined the susceptibilities of Bordetella pertussis strains to several antimicrobial peptides by determining the concentration required to inhibit or kill 50% of the bacterial population. The peptides are ranked in decreasing potency as follows: cecropin B > cecropin A >> melittin > cecropin P1 > (ala8,13,18)-magainin II amide > mastoparan = defensin HNP1 > protamine > or = magainin II = magainin I. By using a radial diffusion assay to compare susceptibilities between strains, wild-type B. pertussis BP338 was more resistant than the avirulent bvg mutant strain BP347 and the brk mutant strain BPM2041 to killing by cecropin P1. In contrast, compared with the wild type, the avirulent BP347 strain was highly resistant to killing by protamine and defensin HNP1.

Effect of antibiotics on Bordetella pertussis adhering activity: hypothesis regarding mechanism of action.

Microbial adherence to epithelial cell surfaces has been implicated as the first step in the initiation of several infectious diseases. The ability of antibiotics to affect the properties of bacterial adherence to cell surfaces may be a criterion in selecting antibiotics for therapy. This study was performed in order to investigate the activity of amoxicillin, chloramphenicol, and clarithromycin in modifying the adhering activity of Bordetella pertussis to human epithelial cells. The actions of antibiotics, alone or combined with aprotinin, were compared with that of trypsin, aprotinin and trypsin+aprotinin, to investigate the chemical nature of the ligand where antibiotics could act. The adhering activity was evaluated on human epithelial cells, collected from the oral mucosa, challenged with B. pertussis A2963 previously incubated in the presence of the tested substances for 1 h at 37 degrees C in a shaker incubator. After staining, the percentage of mucosal cells with more than 50 adhering bacteria was evaluated. Under the described experimental conditions, trypsin significantly reduced the adherence of B. pertussis. Aprotinin had no effect but was able to counteract the inhibitory action of trypsin. Both clarithromycin and chloramphenicol markedly reduced adhering activity and their actions were not counteracted by aprotinin. Amoxicillin was without effect. It was hypothesized that chloramphenicol and clarithromycin, exerting their antimicrobial action by inhibiting bacterial protein synthesis, affected bacterial adhesion through an unknown mechanism without proteolytic effect.