Comparative genomics of the classical Bordetella subspecies: the evolution and exchange of virulence-associated diversity amongst closely related pathogens.

BACKGROUND: The classical Bordetella subspecies are phylogenetically closely related, yet differ in some of the most interesting and important characteristics of pathogens, such as host range, virulence and persistence. The compelling picture from previous comparisons of the three sequenced genomes was of genome degradation, with substantial loss of genome content (up to 24%) associated with adaptation to humans. RESULTS: For a more comprehensive picture of lineage evolution, we employed comparative genomic and phylogenomic analyses using seven additional diverse, newly sequenced Bordetella isolates. Genome-wide single nucleotide polymorphism (SNP) analysis supports a reevaluation of the phylogenetic relationships between the classical Bordetella subspecies, and suggests a closer link between ovine and human B. parapertussis lineages than has been previously proposed. Comparative analyses of genome content revealed that only 50% of the pan-genome is conserved in all strains, reflecting substantial diversity of genome content in these closely related pathogens that may relate to their different host ranges, virulence and persistence characteristics. Strikingly, these analyses suggest possible horizontal gene transfer (HGT) events in multiple loci encoding virulence factors, including O-antigen and pertussis toxin (Ptx). Segments of the pertussis toxin locus (ptx) and its secretion system locus (ptl) appear to have been acquired by the classical Bordetella subspecies and are divergent in different lineages, suggesting functional divergence in the classical Bordetellae. CONCLUSIONS: Together, these observations, especially in key virulence factors, reveal that multiple mechanisms, such as point mutations, gain or loss of genes, as well as HGTs, contribute to the substantial phenotypic diversity of these versatile subspecies in various hosts.

Complete Khoisan and Bantu genomes from southern Africa.

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Microarray and functional analysis of growth phase-dependent gene regulation in Bordetella bronchiseptica.

Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg(+) and Bvg(-) phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system.

Role of the type III secretion system in a hypervirulent lineage of Bordetella bronchiseptica.

Despite the fact that closely related bacteria can cause different levels of disease, the genetic changes that cause some isolates to be more pathogenic than others are generally not well understood. We use a combination of approaches to determine which factors contribute to the increased virulence of a Bordetella bronchiseptica lineage. A strain isolated from a host with B. bronchiseptica-induced disease, strain 1289, was 60-fold more virulent in mice than one isolated from an asymptomatically infected host, strain RB50. Transcriptome analysis and quantitative reverse transcription-PCR showed that the type III secretion system (TTSS) genes were more highly expressed by strain 1289 than strain RB50. Compared to strain RB50, strain 1289 exhibited greater TTSS-mediated cytotoxicity of a mammalian cell line. Additionally, we show that the increase in virulence of strain 1289 compared to that of RB50 was partially attributable to the TTSS. Using multilocus sequence typing, we identified another strain from the same lineage as strain 1289. Similar to strain 1289, we implicate the TTSS in the increased virulence of this strain. Together, our data suggest that the TTSS is involved in the increased virulence of a B. bronchiseptica lineage which appears to be disproportionately associated with disease. These data are consistent with the view that B. bronchiseptica lineages can have different levels of virulence, which may contribute to this species' ability to cause different severities of respiratory disease.

Evidence for horizontal gene transfer of two antigenically distinct O antigens in Bordetella bronchiseptica.

Host immunity is a major driving force of antigenic diversity, resulting in pathogens that can evade immunity induced by closely related strains. Here we show that two Bordetella bronchiseptica strains, RB50 and 1289, express two antigenically distinct O-antigen serotypes (O1 and O2, respectively). When 18 additional B. bronchiseptica strains were serotyped, all were found to express either the O1 or O2 serotype. Comparative genomic hybridization and PCR screening showed that the expression of either the O1 or O2 serotype correlated with the strain containing either the classical or alternative O-antigen locus, respectively. Multilocus sequence typing analysis of 49 B. bronchiseptica strains was used to build a phylogenetic tree, which revealed that the two O-antigen loci did not associate with a particular lineage, evidence that these loci are horizontally transferred between B. bronchiseptica strains. From experiments using mice vaccinated with purified lipopolysaccharide from strain RB50 (O1), 1289 (O2), or RB50Deltawbm (O antigen deficient), our data indicate that these O antigens do not confer cross-protection in vivo. The lack of cross-immunity between O-antigen serotypes appears to contribute to inefficient antibody-mediated clearance between strains. Together, these data are consistent with the idea that the O-antigen loci of B. bronchiseptica are horizontally transferred between strains and encode antigenically distinct serotypes, resulting in inefficient cross-immunity.

Inefficient Toll-like receptor-4 stimulation enables Bordetella parapertussis to avoid host immunity.

The recognition of bacterial lipopolysaccharide (LPS) by host Toll-like receptor (TLR)4 is a crucial step in developing protective immunity against several gram negative bacterial pathogens. Bordetella bronchiseptica and B. pertussis stimulate robust TLR4 responses that are required to control the infection, but a close relative, B. parapertussis, poorly stimulates this receptor, and TLR4 deficiency does not affect its course of infection. This led us to hypothesize that inefficient TLR4 stimulation enables B. parapertussis to evade host immunity. In a mouse model of infection, B. parapertussis grew rapidly in the lungs, but no measurable increase in TLR4-mediated cytokine, chemokine, or leukocyte responses were observed over the first few days of infection. Delivery of a TLR4 stimulant in the inoculum resulted in a robust inflammatory response and a 10- to 100-fold reduction of B. parapertussis numbers. As we have previously shown, B. parapertussis grows efficiently during the first week of infection even in animals passively immunized with antibodies. We show that this evasion of antibody-mediated clearance is dependent on the lack of TLR4 stimulation by B. parapertussis as co-inoculation with a TLR4 agonist resulted in 10,000-fold lower B. parapertussis numbers on day 3 in antibody-treated wild type, but not TLR4-deficient, mice. Together, these results indicate that inefficient TLR4 stimulation by B. parapertussis enables it to avoid host immunity and grow to high numbers in the respiratory tract of naïve and immunized hosts.

Replacement of adenylate cyclase toxin in a lineage of Bordetella bronchiseptica.

Bordetella bronchiseptica is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of B. bronchiseptica strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that cyaA, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the cya locus, which encodes, activates, and secretes CyaA, was replaced by an operon (ptp) predicted to encode peptide transport proteins. Other B. bronchiseptica strains from the same phylogenetic lineage as that of strain 253 also lacked the cya locus, contained the ptp genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the ptp genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of B. bronchiseptica.

Delayed role of tumor necrosis factor- alpha in overcoming the effects of pertussis toxin.

Bordetella pertussis causes whooping cough, an endemic respiratory disease that is increasing in prevalence despite vaccination efforts. Although host immunity is modulated by virulence factors of this pathogen, it is unclear what host factors are required to overcome their effects. Here, we investigate an apparent relationship between the effects of pertussis toxin and tumor necrosis factor (TNF)- alpha . B. pertussis grew efficiently and caused moderate pathology in wild-type mice, whereas TNF- alpha (-/-) mice had higher numbers of bacteria and leukocytes in lungs, experienced more airway resistance, and died of the infection. Interestingly, an isogenic B. pertussis strain lacking pertussis toxin did not induce these effects in TNF- alpha (-/-) mice and behaved similarly in wild-type and TNF- alpha -deficient hosts. Together, these results indicate that TNF- alpha is essential for the host to overcome the effects of pertussis toxin, allowing both control of B. pertussis numbers and regulation of the inflammation induced by infection.