Kimberly A. Kline

Bacterial Adhesins in Host-Microbe Interactions

Most commensal and pathogenic bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that promote interaction with host cell receptors or with soluble macromolecules. Even though bacterial attachment to epithelial cells may be beneficial for bacterial colonization, adhesion may come at a cost because bacterial attachment to immune cells can facilitate phagocytosis and clearing. Many pathogenic bacteria have solved this dilemma by producing an antiphagocytic surface layer usually consisting of polysaccharide and by expressing their adhesins on polymeric structures that extend out from the cell surface. In this review, we will focus on the interaction between bacterial adhesins and the host, with an emphasis on pilus-like structures.

A tale of two pili: assembly and function of pili in bacteria

Bacterial pili have long been recognized as mediators of initial host-pathogen interactions important for the progression of Gram-negative bacterial diseases. An appreciation of the role of pili on virulence in Gram-positive bacteria and the unique properties of their biogenesis is a rapidly emerging area of research. In this review, we focus on recent advances in one of the longest-studied Gram-negative pilus systems, the chaperone/usher assembled pili, along with the newcomer to the field, the sortase-assembled pili of Gram-positive bacteria. In both systems, a wealth of new structural and molecular details has emerged recently. In light of this, we explore similarities between chaperone/usher and sortase-assembled pilus biogenesis and highlight paradigms unique to each, with the goal of using knowledge of each system to raise new questions and inform future studies of the other.

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

ABSTRACT Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.

The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non‐selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac‐regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4 × 10−3 events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non‐piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.

Recombination, repair and replication in the pathogenic Neisseriae: the 3 R′s of molecular genetics of two human‐specific bacterial pathogens

Most of the detailed mechanisms that have been established for the molecular biological processes that mediate recombination, repair and replication of DNA have come from studies of the Escherichia coli paradigm. The human specific pathogens, Neisseria gonorrhoeae and N. meningitidis, are Gram‐negative bacteria that have some molecular processes that are similar to E. coli and others that appear to be divergent. We propose that the pathogenic Neisseriae have evolved a specialized collection of molecular mechanisms to adapt to life limited to human hosts. In this MicroReview, we explore what is known about the basic processes of DNA repair, DNA recombination (genetic exchange and pilin variation) and DNA replication in these human specific pathogens.

G‐CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC‐induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro‐inflammatory cytokines including granulocyte colony‐stimulating factor (G‐CSF, CSF‐3), not previously known to be involved in the host response to UTI. G‐CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G‐CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G‐CSF resulted in a corresponding increase in macrophage‐activating cytokines, such as monocyte chemotactic protein‐1 (MCP‐1, CCL‐2) and Il‐1β, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G‐CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.

Epstein-Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus

Epstein-Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807-2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+ LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.

Combinatorial Small-Molecule Therapy Prevents Uropathogenic Escherichia coli Catheter-Associated Urinary Tract Infections in Mice

ABSTRACT Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens.

Low-Level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria gonorrhoeae

ABSTRACT The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation.

The obligate human pathogen Neisseria gonorrhoeae (Gc) has co‐opted conserved recombination pathways to achieve immune evasion by way of antigenic variation (Av). We show that both the RuvABC and RecG Holliday junction (HJ) processing pathways are required for recombinational repair, each can act during genetic transfer, and both are required for pilin Av. Analysis of double mutants shows that either the RecG or RuvAB HJ processing pathway must be functional for normal growth of Gc when RecA is expressed. HJ processing‐deficient survivors of RecA expression are enriched for non‐piliated bacteria that carry large deletions of the pilE gene. Mutations that prevent pilin variation such as recO, recQ, and a cis‐acting pilE transposon insertion all rescue the RecA‐dependent growth inhibition of a HJ processing‐deficient strain. These results show that pilin Av produces a recombination intermediate that must be processed by either one of the HJ pathways to retain viability, but requires both HJ processing pathways to yield pilin variants. The need for diversity generation through frequent recombination reactions creates a situation where the HJ processing machinery is essential for growth and presents a possible target for novel antimicrobials against gonorrhoea.