Rachel C. Fernandez

A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain

Autotransporter secretion represents a unique mechanism that Gram‐negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa α‐domain and a 30 kDa β domain. The α domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the β domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the β‐domain to ferry the passenger domain to the bacterial surface through a channel formed by the β‐domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601–Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT‐deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601–Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.

Recognition of lipid A variants by the TLR4-MD-2 receptor complex

Lipopolysaccharide (LPS) is a component of the outer membrane of almost all Gram-negative bacteria and consists of lipid A, core sugars, and O-antigen. LPS is recognized by Toll-like receptor 4 (TLR4) and MD-2 on host innate immune cells and can signal to activate the transcription factor NFκB, leading to the production of pro-inflammatory cytokines that initiate and shape the adaptive immune response. Most of what is known about how LPS is recognized by the TLR4-MD-2 receptor complex on animal cells has been studied using Escherichia coli lipid A, which is a strong agonist of TLR4 signaling. Recent work from several groups, including our own, has shown that several important pathogenic bacteria can modify their LPS or lipid A molecules in ways that significantly alter TLR4 signaling to NFκB. Thus, it has been hypothesized that expression of lipid A variants is one mechanism by which pathogens modulate or evade the host immune response. Additionally, several key differences in the amino acid sequences of human and mouse TLR4-MD-2 receptors have been shown to alter the ability to recognize these variations in lipid A, suggesting a host-specific effect on the immune response to these pathogens. In this review, we provide an overview of lipid A variants from several human pathogens, how the basic structure of lipid A is recognized by mouse and human TLR4-MD-2 receptor complexes, as well as how alteration of this pattern affects its recognition by TLR4 and impacts the downstream immune response.

Polar Localization of the Autotransporter Family of Large Bacterial Virulence Proteins

Autotransporters are an extensive family of large secreted virulence-associated proteins of gram-negative bacteria. Secretion of such large proteins poses unique challenges to bacteria. We demonstrate that autotransporters from a wide variety of rod-shaped pathogens, including IcsA and SepA of Shigella flexneri, AIDA-I of diffusely adherent Escherichia coli, and BrkA of Bordetella pertussis, are localized to the bacterial pole. The restriction of autotransporters to the pole is dependent on the presence of a complete lipopolysaccharide (LPS), consistent with known effects of LPS composition on membrane fluidity. Newly synthesized and secreted BrkA is polar even in the presence of truncated LPS, and all autotransporters examined are polar in the cytoplasm prior to secretion. Together, these findings are consistent with autotransporter secretion occurring at the poles of rod-shaped gram-negative organisms. Moreover, NalP, an autotransporter of spherically shaped Neisseria meningitidis contains the molecular information to localize to the pole of Escherichia coli. In N. meningitidis, NalP is secreted at distinct sites around the cell. These data are consistent with a model in which the secretion of large autotransporters occurs via specific conserved pathways located at the poles of rod-shaped bacteria, with profound implications for the underlying physiology of the bacterial cell and the nature of bacterial pathogen-host interactions.

Glucosamine Found as a Substituent of Both Phosphate Groups in Bordetella Lipid A Backbones: Role of a BvgAS-Activated ArnT Ortholog

Endotoxins are amphipathic lipopolysaccharides (LPSs), major constituents of the outer membrane of gram-negative bacteria. They consist of a lipid region, covalently linked to a core oligosaccharide, to which may be linked a repetitive glycosidic chain carrying antigenic determinants. Most of the biological activities of endotoxins have been associated with the lipid moiety of the molecule: unique to gram-negative bacteria, LPS is a ligand of the mammalian TLR4-MD2-CD14 pathogen recognition receptor complex. Lipid A preparations are often heterogeneous with respect to both the numbers and the lengths of fatty acids and the natures of substituents on the phosphate groups when present. The variants can significantly affect host immune responses. Nine species in the Bordetella genus have been described, and the fine LPS structures of seven of them have been published. In this report, lipids A from Bordetella pertussis Tohama I and B. bronchiseptica strain 4650 were further characterized and revealed to have a glucosamine substituting both lipid A phosphate groups of the diglucosamine backbone. These substitutions have not been previously described for bordetellae. Moreover, a B. pertussis transposon mutation that maps within a gene encoding a Bordetella ArnT (formerly PmrK) glycosyl transferase ortholog does not carry this substitution, thus providing a genetic basis for the modification. Reverse transcriptase PCR of this locus showed that it is Bvg regulated, suggesting that the ability of Bordetella to modify lipid A via this glucosamine modification is a potential virulence trait.

Protective activity of the Bordetella pertussis BrkA autotransporter in the murine lung colonization model.

This study examined the vaccine potential of the autotransporter protein BrkA of Bordetella pertussis in the sublethal intranasal murine respiratory challenge model of infection. Five different acellular pertussis (Pa) vaccines, containing different pertussis-component antigens but all comprizing diphtheria (D) and tetanus (T) toxoids, were tested. A two-pertussis-component DTPa vaccine containing pertussis toxoid (PT) and filamentous hemagglutinin (FHA) induced only limited bacterial clearance. However, a three-pertussis-component DTPa vaccine containing PT, FHA and a recombinant BrkA protein (rBrkA) was found to be as efficacious in protecting mice against colonization by B. pertussis strains Tohama I and 18-323 as the commercial Infanrixtrade mark vaccine that also includes PT and FHA but pertactin (PRN) instead of rBrkA. Vaccination of mice with rBrkA as the only B. pertussis antigen did not protect against colonization by B. pertussis. We also demonstrated that BrkA is ubiquitously expressed by highly prevalent clinical isolates of B. pertussis and suggest that new acellular pertussis vaccine formulations that include BrkA have equivalent efficacy as currently available DTPa vaccines against B. pertussis infections.

Bordetella pertussis Binds Human C1 Esterase Inhibitor during the Virulent Phase, to Evade Complement-Mediated Killing

C1 esterase inhibitor (C1inh) is a major inhibitor of several pathways of inflammation in humans. In this study, we show that virulent-phase cultures of Bordetella pertussis, the etiological agent for whooping cough, but not other Bordetella species specifically recruit C1inh from human serum. Using a spontaneous mutant of B. pertussis that was deficient in C1inh binding, we demonstrate that the ability of B. pertussis to acquire high levels of human C1inh and wild-type levels of serum resistance are well correlated, suggesting that, in addition to and independent of BrkA expression, acquisition of C1inh is vital to B. pertussis resistance to complement-mediated killing.

Substitution of the bordetella pertussis lipid a phosphate groups with glucosamine is required for robust nf-κb activation and release of proinflammatory cytokines in cells expressing human but not murine toll-like receptor 4-MD-2-CD14

ABSTRACT Bordetella pertussis endotoxin is a key modulator of the host immune response, mainly due to the role of its lipid A moiety in Toll-like receptor 4 (TLR4)-mediated signaling. We have previously demonstrated that the lipid A phosphate groups of B. pertussis BP338 can be substituted with glucosamine in a BvgAS-regulated manner. Here we examined the effect of this lipid A modification on the biological activity of B. pertussis endotoxin. We compared purified endotoxin and heat-killed B. pertussis BP338 whole cells that have modified lipid A phosphate groups to an isogenic mutant lacking this modification with respect to their capacities to induce the release of inflammatory cytokines by human and murine macrophages and to participate in the TLR4-mediated activation of NF-κB in transfected HEK-293 cells. We found inactivated B. pertussis cells to be stronger inducers of proinflammatory cytokines in THP-1-derived macrophages when lipid A was modified. Most notably, lack of lipid A modification abolished the ability of purified B. pertussis endotoxin to induce the release of inflammatory cytokines by human THP-1-derived macrophages but led to only slightly reduced inflammatory cytokine levels when stimulating murine (RAW 264.7) macrophages. Accordingly, upon stimulation of HEK-293 cells with inactivated bacteria and purified endotoxin, lack of lipid A modification led to impaired NF-κB activation only when human, and not when murine, TLR4-MD-2-CD14 was expressed. We speculate that in B. pertussis, lipid A modification has evolved to benefit the bacteria during human infection by modulating immune defenses rather than to evade innate immune recognition.

Antibodies to BrkA augment killing of Bordetella pertussis

BrkA is a Bvg-regulated Bordetella pertussis protein that mediates serum resistance and adherence. It shares sequence identity with another B. pertussis virulence factor called pertactin, and it is a member of the diverse group of proteins found in Gram-negative bacteria that are secreted by an autotransporter mechanism. Sera, either from individuals who have been vaccinated with acellular pertussis vaccines, or from individuals who have no re-collection of recent infection with B. pertussis fail to kill wild-type B. pertussis, but kill brkA mutant strains very well. We examined whether BrkA could be neutralised in serum fitting this profile. BrkA is synthesised as a 103kDa precursor that is processed into a surface-associated N-terminal 73kDa passenger domain, and an outer-membrane embedded C-terminal 30kDa transporter moiety. Polyclonal antibodies were raised to a recombinant, re-folded histidine-tagged fusion protein representing the 73kDa passenger region. These anti-BrkA antibodies were shown to boost the existing bactericidal capacity of human serum against B. pertussis by neutralising BrkA.

Minor Modifications to the Phosphate Groups and the C3′ Acyl Chain Length of Lipid A in Two Bordetella pertussis Strains, BP338 and 18-323, Independently Affect Toll-like Receptor 4 Protein Activation

Background: Lipid A activation of TLR4 shapes immunity to Gram-negative bacteria. Results: In Bordetella pertussis lipid A, the genetic basis for longer C3′ acyl chains and glucosamine modification of the phosphate groups was identified; each variation independently increased TLR4 activation. Conclusion: Minor changes in the penta-acylated B. pertussis lipid A affect TLR4 activation. Significance: This aids our understanding how lipid A species interact with TLR4. Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4′-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3′ acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399–BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3′ acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.

Identification and characterization of autotransporter proteins of Yersinia pestis KIM.

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.