Browsing by Author "St. Geme III, Joseph W"

Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal β-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.

Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the β-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,

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we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.

The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.

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In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.

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Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most β-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.

In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.

Disease caused by the Gram-negative Haemophilus cryptic genospecies begins with colonization of the maternal genital or neonatal respiratory tract. The primary goal of this work was to identify and characterize the molecular determinant(s) of Haemophilus cryptic genospecies adherence as a means to better understand the specific adaptation of this species to the urogenital tract and neonatal respiratory tract. Using transposon mutagenesis of prototype strain 1595, we identified a locus that is essential for Haemophilus cryptic genospecies adherence to a variety of epithelial cell lines of both genital and respiratory origin. This locus encodes a protein called Cha that shares homology with trimeric autotransporters. Trimeric autotransporters are composed of an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal membrane anchor domain and are classically characterized by head-stalk-anchor domain architecture. By generating chimeric proteins, we demonstrated that the C-terminus of Cha trimerizes in the bacterial outer membrane and is capable presenting a heterologous passenger domain (Hia) in a functional form, thus confirming that Cha is a trimeric autotransporter. Southern analysis revealed that cha is unique to the Haemophilus cryptic genospecies and is ubiquitous among these strains.

Similar to a number of trimeric autotransporters, the passenger domain of Cha contains scattered clusters of YadA-like head domains associated with head-to-stalk neck adaptor motifs, predicted coiled-coil stalks and a series of identical tandem coding repeats which are not required for adherence. By evaluating the adherence capacity of H. influenzae expressing Cha deletion derivatives, we established that the N-terminal 473 residues of Cha harbor the binding domains responsible for Cha-mediated adherence to epithelial cells. In additional studies, we demonstrated that this same N-terminal region mediates bacterial aggregation through inter-bacterial Cha-Cha binding.

Further analysis revealed that variable Cha-mediated adherence is linked to spontaneous changes in the number of identical tandem repeats predicted to comprise a coiled-coil stalk domain. Variation in repeat copy number has a direct effect on Cha adhesive and aggregative activity, independent of an impact on transcription of the cha locus or surface localization of Cha protein. Moreover, length of Cha surface fibers correlates with repeat copy number expansion. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats may influence the conformation of Cha, thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface that may have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells.

In additional studies screening adherent cryptic genospecies isolates for expression of Cha protein, we identified an additional, antigenically-divergent Cha variant that we refer to as Cha2. Amino acid sequence and domain comparison of Cha2 with Cha (now Cha1) revealed that the structurally undefined N-terminal sequences (encompassing the Cha1 adhesive and aggregative domain) are strikingly divergent. Inspite of this, Cha2 mediates efficient adherence to human epithelial cells, similar to Cha1.

Identification of Cha offers insight into the apparent tissue tropism associated with the Haemophilus cryptic genospecies. We speculate that the unique regulation of Cha adhesive activity enhances the adaptive capability of this pathogenic organism in the human host.

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium in the posterior pharynx. Previous work identified type IV pili as a critical factor for adherence to human epithelial cells. However, the finding that a significant percentage of pharyngeal isolates are non-piliated suggests that K. kingae expresses additional surface factors that modulate interactions with host cells and likely play key roles in the pathogenesis of K. kingae disease. The purpose of this work was to increase our understanding of K. kingae virulence determinants, specifically focused on defining the surface factors and the mechanism involved in K. kingae adhesive interactions with epithelial cells. Additionally, this work aimed to further characterize components of the K. kingae type IV pilus system, namely the PilC proteins and PilA2.

We first set out to identify non-pilus factors that influence K. kingae interactions with human epithelial cells. Using targeted genetic approaches, we found that insertional inactivation of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, using a variety of techniques, including morphological analysis, cationic ferritin staining, and thin section transmission electron microscopy, we established that K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called ctrABCD for surface presentation. Furthermore, using quantitative human epithelial cell adherence assays, we discovered that the presence of surface capsule interferes with Knh-mediated adherence by non-piliated organisms and that maximal adherence in the presence of capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. Based on the data presented here, we propose a novel adherence mechanism that allows K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance.

Having established that K. kingae produces a capsule, a large-scale polysaccharide purification technique was developed for capsule analysis of strain 269-492. Biochemical assays determined that the purified material contained thiobarbituric and phenol-sulfuric acid reactive glycosyl residues. In collaboration with the University of Georgia Complex Carbohydrate Research Center (CCRC), mass spectrometry identified galactose, N-acetyl-galactosamine, and Kdo as the major glycosyl components of the polysaccharide preparation. NMR spectroscopy revealed that the purified material contained two distinct polysaccharides with the structures of →5)–β–Galf–(1→ and →3)–β–GalNAcp–(1→5)–β–Kdop–(2→. Further characterization of the polysaccharides expressed by K. kingae may have implications for disease prevention strategies.

Previous work in our lab found that two PilC-like proteins called PilC1 and PilC2 influence type IV pili expression and pilus-mediated adherence. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. We set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional nine amino acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12 amino acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we show that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.

Lastly, we set out to define the role of the PilA2 minor pilin in K. kingae strain 269-492. While previous studies indicated that PilA2 is not essential for pilus expression or adherence to epithelial cells, analysis of the pilin locus in a diverse set of clinical isolates revealed that the pilA2 gene sequence is highly conserved, suggesting it serves an important function. Using targeted mutagenesis we showed that PilA2 is not essential for twitching motility and may or may not be involved in natural competence. Western blot analysis was unable to detect PilA2 in wild type pilus preparations, indicating that it is expressed at a level beneath the assay detection limit or does not localize to the pilus. Additionally, site-directed mutagenesis was used to place pilA2 under control of the highly active pilA1 promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells.

Taken together, this work expands our knowledge of the K. kingae surface factor repertoire and provides insights into the roles of type IV pilus components. The mechanism of K. kingae adherence to epithelial cells is beginning to emerge. These contributions may lead to novel strategies for the prevention of invasive K. kingae disease in young children.