Steven Clegg

Differential binding to and biofilm formation on, HEp‐2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster

Type 1 fimbria‐mediated adherence to HEp‐2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose‐sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp‐2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp‐2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp‐2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp‐2 cells using once‐flow‐through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.

Utilization of an Intracellular Bacterial Community Pathway in Klebsiella pneumoniae Urinary Tract Infection and the Effects of FimK on Type 1 Pilus Expression

ABSTRACT Klebsiella pneumoniae is an important cause of urinary tract infection (UTI), but little is known about its pathogenesis in vivo. The pathogenesis of the K. pneumoniae cystitis isolate TOP52 was compared to that of the uropathogenic Escherichia coli (UPEC) isolate UTI89 in a murine cystitis model. Bladder and kidney titers of TOP52 were lower than those of UTI89 at early time points but similar at later time points. TOP52, like UTI89, formed biofilm-like intracellular bacterial communities (IBCs) within the murine bladder, albeit at significantly lower levels than UTI89. Additionally, filamentation of TOP52 was observed, a process critical for UTI89 evasion of neutrophil phagocytosis and persistence in the bladder. Thus, the IBC pathway is not specific to UPEC alone. We investigated if differences in type 1 pilus expression may explain TOP52s early defect in vivo. The type 1 pilus operon is controlled by recombinase-mediated (fimE, fimB, and fimX) phase variation of an invertible promoter element. We found that K. pneumoniae carries an extra gene of unknown function at the 3′ end of its type 1 operon, fimK, and the genome lacks the recombinase fimX. A deletion mutant of fimK was constructed, and TOP52 ΔfimK had higher titers and formed more IBCs in the murine cystitis model than wild type. The loss of fimK or expression of E. coli fimX from a plasmid in TOP52 resulted in a larger phase-ON population and higher expression levels of type 1 pili and gave TOP52 the ability to form type 1-dependent biofilms. Complementation with pfimK decreased type 1 pilus expression and biofilm formation of TOP52 ΔfimK and decreased UTI89 biofilm formation. Thus, K. pneumoniae appears programmed for minimal expression of type 1 pili, which may explain, in part, why K. pneumoniae is a less prevalent etiologic agent of UTI than UPEC.

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue‐binding specificity of the type‐3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild‐type Kleb‐siella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type‐3 fimbriae, as well as the purified type‐3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowmans capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type‐3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type‐3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type‐3 fimbrial gene cluster in association with the pap‐encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type‐3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type‐3 fimbriae strains of Yeisinia and Salmonella, showing that the ability to use type collagen as tissue target is widespread among enteric bacteria.

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type‐1‐fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type‐1‐fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non‐fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside‐inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae‐coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type‐1 ‐fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type‐1‐fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type‐1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type‐1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.

Type 3 Fimbrial Shaft (MrkA) of Klebsiella pneumoniae, but Not the Fimbrial Adhesin (MrkD), Facilitates Biofilm Formation

ABSTRACT Isolates of Klebsiella pneumoniae are responsible for opportunistic infections, particularly of the urinary tract and respiratory tract, in humans. These bacteria express type 3 fimbriae that have been implicated in binding to eucaryotic cells and matrix proteins. The type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. The formation of biofilms in vitro by strains ofK. pneumoniae was shown to be affected by the production of fimbriae on the bacterial surface. However, a functional MrkD adhesin was not necessary for efficient biofilm formation. Nonfimbriate strains were impaired in their ability to form biofilms. Using isogenic fimbriate and nonfimbriate strains of K. pneumoniaeexpressing green fluorescent protein it was possible to demonstrate that the presence of type 3 fimbriae facilitated the formation of dense biofilms in a continuous-flowthrough chamber. Transformation of nonfimbriate mutants with a plasmid possessing an intactmrk gene cluster restored the fimbrial phenotype and the rapid ability to form biofilms.

Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

ABSTRACT Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose‐containing glycoconjugates, is the conversion of mannose‐6‐phosphate to mannose‐1‐phosphate, by a phosphomannomutase (PMM), to produce GDP‐mannose, the primary mannose‐donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 ± 3.9‐fold greater association of mycobacteria with human macrophages, in a mannan‐inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.

FimW is a negative regulator affecting type 1 fimbrial expression in Salmonella enterica serovar typhimurium.

Type 1 fimbriae are proteinaceous surface appendages that carry adhesins specific for mannosylated glycoproteins. These fimbriae are found on most members of the family Enterobacteriaceae and are known to facilitate binding to a variety of eukaryotic cells, including those found on the mucosal surfaces of the alimentary tract. We have shown that the regulation of type 1 fimbrial expression in Salmonella enterica serovar Typhimurium is controlled, in part, by the products of four genes found within the fim gene cluster: fimZ, fimY, fimW, and fimU. To better understand the specific role of FimW in fimbrial expression, a mutation was constructed in this gene by the insertion of a kanamycin resistance DNA cassette into the chromosome. The resulting fimW mutation was characterized by mannose-sensitive hemagglutination and agglutination with fimbria-specific antiserum. Assays suggested that this mutant was more strongly fimbriate than the parental strain, exhibiting a four- to eightfold increase in fimbrial production. The fimW mutation was introduced into a second strain of Salmonella enterica serovar Typhimurium, and this mutant was also found to be strongly fimbriate compared to the parental strain. Consistent with the role of this protein as a negative regulator, fimA-lacZ expression in serovar Typhimurium, as well as in Escherichia coli, was increased twofold in the absence of functional FimW. Primer extension analysis determined that fimW transcription is initiated from its own promoter 31 bp upstream of the translation start site. Analysis using a fimW-lacZ reporter indicated that fimW expression in serovar Typhimurium was increased under conditions that select for poorly fimbriate bacteria and low fimA expression. FimW also appears to act as an autoregulator, since expression from the fimW-lacZ reporter was increased in a fimW mutant. FimW was partially purified by fusion with the E. coli maltose-binding protein. Use of this FimW protein extract, as well as others, in DNA-binding assays was unable to identify a specific binding site for FimW in the fimA, fimZ, fimY, or fimW promoter regions. To analyze protein-protein interactions, FimW was expressed in a LexA-based two-hybrid system in E. coli. A significant interaction between FimW and the DNA-binding activator protein, FimZ, was detected using this system. These results indicate that FimW is a negative regulator of serovar Typhimurium type 1 fimbrial expression and may function by interfering with FimZ-mediated activation of fimA expression.

Klebsiella pneumoniae and type 3 fimbriae: nosocomial infection, regulation and biofilm formation

The Gram-negative opportunistic pathogen Klebsiella pneumoniae is responsible for causing a spectrum of nosocomial and community-acquired infections. Globally, K. pneumoniae is a frequently encountered hospital-acquired opportunistic pathogen that typically infects patients with indwelling medical devices. Biofilm formation on these devices is important in the pathogenesis of these bacteria, and in K. pneumoniae, type 3 fimbriae have been identified as appendages mediating the formation of biofilms on biotic and abiotic surfaces. The factors influencing the regulation of type 3 fimbrial gene expression are largely unknown but recent investigations have indicated that gene expression is regulated, at least in part, by the intracellular levels of cyclic di-GMP. In this review, we have highlighted the recent studies that have worked to elucidate the mechanism by which type 3 fimbrial expression is controlled and the studies that have established the importance of type 3 fimbriae for biofilm formation and nosocomial infection by K. pneumoniae.

Salmonella enterica Serovars Gallinarum and Pullorum Expressing Salmonella enterica Serovar Typhimurium Type 1 Fimbriae Exhibit Increased Invasiveness for Mammalian Cells

ABSTRACT Salmonella enterica serovars Gallinarum and Pullorum are S. enterica biotypes that exhibit host specificity for poultry and aquatic birds and are not normally capable of causing disease in mammalian hosts. During their evolution toward host restriction serovars Gallinarum and Pullorum lost their ability to mediate mannose-sensitive hemagglutination (MSHA), a phenotype correlated with adherence to certain cell types. Because adherence is an essential requirement for invasion of cells by bacterial pathogens, we examined whether MHSA type 1 fimbriae would increase the ability of serovars Pullorum and Gallinarum to invade normally restrictive cells. Serovars Gallinarum and Pullorum expressing S. entericaserovar Typhimurium strain LT2 type 1 fimbriae exhibited a 10- to 20-fold increased ability to adhere to and a 20- to 60-fold increased invasion efficiency of the human epithelial HEp-2 cell line. Invasion was accompanied by extensive ruffling of the membranes of the HEp-2 cells. In a murine ligated ileal loop model, a 32% increase in the number of M-cell ruffles was seen when serovar Gallinarum expressed serovar Typhimurium type 1 fimbriae.