Carbohydrate receptors of bacterial adhesins: implications and reflections.

Bacteria entering a host depend on adhesins to achieve colonization. Adhesins are bacterial surface structures mediating binding to host surficial areas. Most adhesins are composed of one or several proteins. Usually a single bacterial strain is able to express various adhesins. The adhesion type expressed may influence host-, tissue or even cell tropism of Gram-negative and of Gram-positive bacteria. The binding of fimbrial as well as of afimbrial adhesins of Gram-negative bacteria to host carbohydrate structures (=receptors) has been elucidated in great detail. In contrast, in Gram-positives, most well studied adhesins bind to proteinaceous partners. Nevertheless, for both bacterial groups the binding of bacterial adhesins to eukaryotic carbohydrate receptors is essential for establishing colonization or infection. The characterization of this interaction down to the submolecular level provides the basis for strategies to interfere with this early step of infection which should lead to the prevention of subsequent disease. However, this goal will not be achieved easily because bacterial adherence is not a monocausal event but rather mediated by a variety of adhesins.

[Recurrent urinary tract infections in women. Virulence of pathogens and host reaction]. / Rezidivierende Harnweginfektionen der Frau. Erregervirulenz und Wirtsreaktion.

The interactions of host-specific and microbial factors are responsible for recurrent urinary tract infections (rUTI). The anatomical properties of the female urogenital tract favor colonization by uropathogens. Local factors such as glucose concentration of the urine, stability of the lactobacilli population, the influence of estrogens, the activity of Tamm-Horsfall protein or of defensins, and disturbance of the systemic defense mechanisms dictate the course of an infection. The most prominent uropathogen is E. coli. It expresses various virulence factors including adhesions, toxins, iron uptake systems, and a capsule. It is still unclear if there are virulence factors characteristic for E. coli strains causing rUTI. The formation of intracellular reservoirs by invasion of uroepithelial cells by E. coli could be another, as yet little noticed cause for rUTI. Therefore, in cases of rUTI the application of intracellularly active antibiotics should be considered.

Bacterial response to eukaryotic cells. Analysis of differentially expressed proteins using nano liquid chromatography-electrospray ionization tandem mass spectrometry.

Host-bacteria interactions have mostly been investigated with regard to the host response or to activities of pathogenic bacteria. In contrast, we aim to identify reactions of non-pathogenic bacteria that result from their contact with host cells of the gastrointestinal tract. In a proteomic approach, the response of non-pathogenic human Escherichia coli bacteria on gut epithelial cells (rat IEC-6) was investigated in an in vitro co-culture model. For this purpose, a sensitive analytical procedure was developed based on the identification of two-dimensional polyacrylamide gel electrophoresis separated proteins by online nanoLC-electrospray ionization MS/MS using a quadrupole time-of-flight tandem mass spectrometer for accurate mass determination. We demonstrate here the efficiency of this technique by the identification of a total of 43 differentially expressed proteins, out of which 25 were up-regulated and 18 were down-regulated. They represent a wide range of molecular weight and different metabolic and physiological functions.

The high-pathogenicity island is absent in human pathogens of Salmonella enterica subspecies I but present in isolates of subspecies III and VI.

In this study we tested 74 Salmonella strains of all eight Salmonella groups and were able to demonstrate the presence of two high-pathogenicity island types in strains of Salmonella groups IIIa, IIIb, and VI. Most high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were positive for the high-molecular-weight proteins HMWP1 and HMWP2.

Pathogenicity islands of uropathogenic E. coli and the evolution of virulence.

Uropathogenic Escherichia coli (UPEC) are the most important group of microorganisms responsible for urinary tract infection. UPEC differ from non-pathogenic E. coli and from other E. coli pathotypes by the production of specific virulence factors, which enable the bacteria to adhere to uroepithelial cells and to establish urinary tract infections. Besides adherence factors, toxins, 'modulins', capsules, iron uptake systems and other bacterial products contribute to the virulence of the strains. The respective genes are frequently located on large pieces of DNA called 'pathogenicity islands' (PAIs). PAIs form (unstable) regions of the genome of UPECs, which are often associated with tRNA genes. Using various molecular techniques, the functions of PAI encoded gene products have been studied. The usage of DNA arrays give answers to questions on the distribution of PAIs among various enterobacteria and on the expression of the different genes under in vitro and in vivo conditions. In addition, assumptions can be made on the evolution of these important pathogens.

Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts.

High pathogenicity islands (HPIs), first identified in various Yersinia species, encode an iron uptake system. We have studied the occurrence of HPIs in septicemic strains of Escherichia coli isolated from a variety of hosts. The results presented in this communication indicate that most septicemic strains tested contained HPI sequences even though they already have the aerobactin encoding genes. We have also observed two types of HPI deletions, suggesting genetic instability of this element. Notable exceptions are several strains isolated from septicemia in sheep that lacked both iron acquisition systems.

Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae.

The adhesion of K21a, K26, K36, and K50 capsulated Klebsiella strains to ileocecal (HCT-8) and bladder (T24) epithelial cell lines was significantly lower than that of their corresponding spontaneous noncapsulated variants K21a/3, K26/1, K36/3, and K50/3, respectively. Internalization of the bacteria by both epithelial cell lines was also significantly reduced. Similarly, a capsule-switched derivative, K2(K36), that exhibited a morphologically larger K36 capsule and formed more capsular material invaded the ileocecal epithelial cell line poorly compared to the corresponding K2 parent strain. None of the capsulated strains exhibited significant mannose-sensitive type 1 fimbriae, whereas two of the noncapsulated variants K21a/3 and K50/3 exhibited potent mannose-sensitive hemagglutinating activity. Although hemagglutinating activity that could be attributed to mannose-resistant Klebsiella type 3 fimbriae was weak in all strains, in several cases the encapsulated parent strains exhibited lower titers than their corresponding noncapsulated variants. Although the level of adhesion to the ileocecal cells is not different from adhesion to bladder cells, bacterial internalization by bladder cells was significantly lower than internalization by ileocecal cells, suggesting that bladder cells lack components required for the internalization of Klebsiella.

Receptor structure for F1C fimbriae of uropathogenic Escherichia coli.

F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), beta1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc(4)Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM(2) [GgO(3)Cer]) and gangliotetraosylceramide (asialo-GM(1) [GgO(4)Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM(2) (GgO(3)Cer). The bacterial interaction with asialo-GM(1) (GgO(4)Cer) and asialo-GM(2) (GgO(3)Cer) was strongly inhibited only by disaccharide GalNAcbeta1-4Galbeta linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb(5)Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcbeta1-4Galbeta is linked to glucosylceramide as in asialo-GM(2) (GgO(3)Cer). Thus, it is suggested that the disaccharide sequence GalNAcbeta1-4Galbeta of asialo-GM(2) (GgO(3)Cer) which is positioned internally in asialo-GM(1) (GgO(4)Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.

[Adherence and invasion-two pathogenicity factors in bacterial and fungal pathogens]. / Adhärenz und Invasion: Zwei Pathogenitätsfaktoren bei pathogenen Bakterien und Pilzen.

Pathogenicity factors such as adhesins, toxins, capsules, and other microbial gene products are involved as causative agents for infectious diseases. Therefore, the pathogenicity of organisms is increasingly studied on a molecular level. In bacteriology, unspecific adherence mechanisms and receptor-specific adhesins have to be distinguished. An adhesin-mediated invasion of pathogenic organisms in eukaryotic host cells could be relevant for pathogenesis. In mycology, various specific adhesins are involved in colonization of the host. Aspartyl proteases and phospholipases are relevant for adherence and invasion of host structures by pathogenic yeasts. Resistance factors have a central function in the distribution of infectious organisms. Gene-transfer, point mutations and efflux mechanisms are involved in the development of antibiotic drug resistance. Antifungal drug resistance does occur predominantly in Candida albicans against azole drugs. As underlying mechanisms point mutations in the ERG11 gene, encoding for the target enzyme of azoles, as well as energy-dependent efflux mechanisms were identified. Whether these mycotic factors are specific virulence factors or "fitness-factors" for a better survival of these organisms in the host, and if a possible alternating effect exists between resistance and virulence mechanisms is currently under investigation.

N-glycosylated proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured human epithelial cells.

Klebsiella pneumoniae obtained from patients with urinary tract infections is able to invade cultured human epithelial cells. The internalization process is dependent upon both microfilaments and microtubules. To better understand the interaction of these invasive bacteria with the host cell receptor(s), bladder, lung, and ileocecal epithelial cells were infected with K. pneumoniae in the presence of various lectins possessing multiple glycan specificities. It was found that the N-acetylglucosamine (GlcNAc)-specific lectins concanavalin A, Datura stramonium agglutinin, and wheat germ agglutinin significantly inhibited the invasion of K. pneumoniae into these cells but did not interfere with the internalization of an invasive strain of Salmonella typhimurium. Conversely, internalization of K. pneumoniae but not S. typhimurium was also significantly inhibited when the bacteria were pretreated with GlcNAc or chitin hydrolysate, a GlcNAc polymer, prior to the gentamicin invasion assay. Other carbohydrates such as glucose, galactose, mannose, fucose, and N-acetylneuraminic acid had no inhibitory effects on K. pneumoniae uptake. Furthermore, internalization of K. pneumoniae but not S. typhimurium by HCT8 cells was also significantly inhibited when eukaryotic protein glycosylation was interrupted by tunicamycin or when host N-linked surface glycans were removed by pretreatment with N-glycosidase F. These studies suggest that a N-glycosylated protein receptor is involved in the internalization of K. pneumoniae by human epithelial cells in vitro. The results also indicate that internal GlcNAc residues might be a carbohydrate component of the receptor.

Invasion of cultured human epithelial cells by Klebsiella pneumoniae isolated from the urinary tract.

The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091, isolated from a urine sample of a patient with a urinary tract infection, invaded human epithelial cells from the bladder and ileocecum and persisted for days in vitro. Electron microscopic studies demonstrated that K. pneumoniae was always contained in endosomes. The internalization mechanism(s) triggered by K. pneumoniae was studied by invasion assays conducted with different inhibitors that act on prokaryotic and eukaryotic cell structures and processes. Chloramphenicol inhibition of bacterial uptake revealed that bacterial de novo protein synthesis was essential for efficient invasion by K. pneumoniae and S. typhi. Interference with receptor-mediated endocytosis by g-strophanthin or monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular K. pneumoniae cells, but not S. typhi cells. The depolymerization of microfilaments by cytochalasin D inhibited the uptake of both bacteria. Microtubule depolymerization caused by colchicine, demecolcine, or nocodazole and the stabilization of microtubules with taxol reduced only the invasion ability of K. pneumoniae. S. typhi invasion was unaffected by microtubule depolymerization or stabilization. These data suggest that the internalization mechanism triggered by K. pneumoniae 3091 is strikingly different from the solely microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria, such as enteroinvasive Escherichia coli, Salmonella, Shigella, and Yersinia strains.

Uptake pathways of clinical isolates of Proteus mirabilis into human epithelial cell lines.

Proteus mirabilis isolates obtained from urine and faeces showed high invasion levels into several human epithelial cell lines in gentamicin assays. Invasion efficiencies of isolate 102 from a monkey with diarrhoea equalled or even exceeded those of Salmonella typhi strain Ty2 (6.3 to 13.8% of the inoculum). Vegetative, non-swarming P.mirabilis invaded epithelial cells efficiently and were found in endosomes and free in the cytoplasm. Although inhibition of eukaryotic protein synthesis by cycloheximide did not reduce bacterial uptake, inhibition with bacteriostatic antibiotics of bacterial protein-, RNA-, or DNA-synthesis reduced invasion drastically. Involvement of eukaryotic structures and processes in internalization was determined by using various inhibitors in the invasion assay. Uptake of P.mirabilis isolated from urine into gut (INT 407, HCT-8) cells and bladder (T24) cells was dramatically inhibited only by microfilament depolymerization. Internalization of faecal isolate 102 into gut or bladder epithelial cells was inhibited by depolymerization of microfilaments or microtubules. Engulfment of isolate 102 into T24 bladder cells was also reduced by inhibition of receptor-mediated endocytosis. Interference with endosome acidification decreased the number of intracellular bacteria of isolate 102 in all three cell lines. These results suggest that P.mirabilis isolates from different sources are internalized by epithelial cells by different eukaryotic processes, and that these processes can vary between cell lines.

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.

Some structures and processes of human epithelial cells involved in uptake of enterohemorrhagic Escherichia coli O157:H7 strains.

Several enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 isolated from patients with hemorrhagic colitis, ischemic colitis, or hemolytic uremic syndrome were all found to be able to invade certain human epithelial cell lines in vitro. Their ability to gain entry into epithelial cells was compared with those of known invasive Shigella flexneri and Salmonella typhi strains and the noninvasive E. coli strain HB101 in invasion assays utilizing gentamicin to kill extracellular bacteria. All EHEC strains under investigation were efficiently internalized into T24 bladder and HCT-8 ileocecal cells. In striking contrast to shigellae, the same EHEC strains were not taken up into human embryonic intestinal INT407 cells or HEp-2 cells any more than the noninvasive E. coli strain HB101. The mechanism(s) of EHEC internalization was characterized by comparing the invasion efficiencies in the absence and presence of a variety of inhibitors acting on structures and processes of prokaryotic or eukaryotic cells. Also, wild-type, plasmid-containing EHEC strains were compared with their plasmid-cured isogenic derivative strains to determine if plasmid genes affect invasion ability. Plasmid-cured EHEC invaded as well as wild-type EHEC, indicating that invasion ability is chromosomally encoded. Inhibition of bacterial protein synthesis by simultaneous addition of bacteria and chloramphenicol to the monolayer blocked EHEC uptake dramatically, suggesting the presence of an invasion protein(s) with a short half-life. Studies utilizing inhibitors which act on eukaryotic cells demonstrated a strong dependence on microfilaments in the process of uptake of all EHEC strains into both T24 and HCT-8 cells. In general, depolymerization of microtubules as well as inhibition of receptor-mediated endocytosis reduced the efficiency of EHEC invasion of T24 cells, whereas interference with endosome acidification reduced EHEC entry into only HCT-8 cells. Taxol-induced stabilization of microtubules did not inhibit internalization into T24 cells or into the HCT-8 cell line. In marked contrast, the ability of S. typhi Ty2 to invade either cell line was inhibited only by depolymerization of microfilaments. In addition to the cell line specificity of EHEC invasion, not all EHEC strains displayed uniform behavior in the presence of inhibitors, suggesting the existence of variant uptake pathways in different strains. Most importantly, previous reports of the inability of EHEC to invade INT407 or HEp-2 cell lines support the currently held belief that EHEC strains are noninvasive.(ABSTRACT TRUNCATED AT 400 WORDS)

Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii.

Bacterial invasion of six different human epithelial cell lines showed that some strains of the intestinal pathogen Campylobacter jejuni invaded intestinal cell lines at a level 10(2)-10(4) times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacter jejuni strain 81-176 uptake into 407 intestinal cells and Citrobacter entry into T24 bladder cells was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microfilament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacter jejuni or Citrobacter freundii, but monensin markedly reduced Citrobacter uptake. Epithelial cell invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cell protein synthesis. In contrast to the T24 cell line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule- and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfilament-dependent) that are cell line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.