Overview

Our laboratory is interested in bacterial pathogenesis.  We primarily study interactions between pathogenic Escherichia coli and host cells.  Although most E. coli strains are harmless, some are endowed with the capacity to cause diarrhea or systemic illnesses.  Our lab concentrates on a type of E. coli known as enteropathogenic E. coli or EPEC , which causes severe diarrhea in infants, and on uropathogenic E. coli , which cause urinary tract infections.  We use combined molecular biology, biochemisty and cell biology approaches to gain insight into the processes by which pathogenic E. coli cause disease.  The goal is to identify and characterize bacterial factors that are involved in interactions between the pathogens and host cells and to identify cellular mechanisms triggered by these interactions.  Our initial approach, to identify clone and mutate bacterial genes involved in pathogenesis has led to the characterization of large gene clusters and pathogenicity islands required for adherence and host cell damage.  We are currently involved in delineating the functions of the protein products of these genes as well as examining cells for changes induced by infection with bacteria. It is anticipated that these studies might impact not only our understanding and approach to infectious diseases, but also our appreciation for normal and abnormal cellular physiology. 

Definition and Epidemiology

    EPEC are strains of E. coli capable of destroying the microvilli of host cells and inducing the formation of cup-like actin-rich pedestals to which the bacteria intimately attach by a process known as attaching and effacing .  Among the attaching and effacing E. coli, EPEC are distinguished from enterohemorrhagic E. coli by the fact that EPEC do not produce Shiga toxins.  Typical EPEC also possess a large plasmid that mediates the ability of these strains to adhere to host cells in a pattern known as localized adherence .  Atypical EPEC strains lack this plasmid and do not perform localized adherence.

    EPEC strains are a leading cause of diarrhea in developing countries throughout the world.  In some locations EPEC is the most common bacterial cause of diarrhea in infants.  Disease due to EPEC is rare in developed countries.  In poorer countries where EPEC is prevelant it causes disease almost exclusively in infants, especially those under 6 months of age.  Diarrhea due to EPEC is frequently accompanied by vomiting, making oral rehydration difficult.  In addition, EPEC is one of the few recognized causes of protracted diarrhea.  Thus, EPEC is an important human pathogen on a global scale.

Localized Adherence and the Bundle-forming Pilus

    When typical EPEC strains adhere to tissue culture cells or to the intestinal epithelium of infants, they do not cover the entire surface of the cells.  Instead they form three dimensional microcolonies, a pattern known as localized adherence.  The ability to form these microcolonies is conferred by a large plasmid common to typical EPEC strains and, in particular, by a cluster of 14 genes known as the bfp operon .  The bfp operon is sufficient, when expressed under the control of an artificial promoter in a recombinant E. coli strain, to direct the synthesis of a surface appendage known as the bundle-forming pilus [23] , so named because of its tendancy to aggregate into rope-like bundles.  The bundle-forming pilus is a member of a family of pili produced by important pathogens of humans and domestic animals known as Type IV fimbriae.   The mechanisms by which type IV fimbriae are synthesized, exported, assembled, and function are largely obscure.  Our hypothesis states that ten of the fourteen bfp genes together comprise a single molecular machine that carries out these functions.  One of the goals of the laboratory is to describe the architecture of this machine so that a better understanding of the biogenesis of type IV fimbriae may emerge.  We are progressing toward this goal by describing the location and topology of each protein [51 , 53 ], purifying the proteins (Fig. 7 ) and examining interactions among them.
 
 

Attaching and Effacing and the Esp Secreted Proteins

    The hallmark of EPEC infection is the destruction of host cell microvilli and the formation of cup-like pedestals to which the bacteria attach .  These pedestals are composed of host cytoskeletal proteins such as actin.  All of the genes required to cause this attaching and effacing effect are located on a contiguous block of DNA known as the Locus of Enterocyte Effacement (LEE) pathogenicity island [33] .  The sequence of the LEE suggests that it arose in a species other than E. coli and was transferred to certain E. coli strains as a unit.  The LEE consists of 41 genes that can be divided into several groups .  Many of the genes located at one side of the LEE encode components of a type III secretion system.  These systems, common to several important pathogenic Gram-negative bacteria, direct the export of critical virulence determinants out of the bacteria and the import of some of these proteins into the host cell.  Members of the Kaper lab are currently investingating the EPEC type III secretion apparatus.  At the other end of the LEE are genes that encode proteins secreted by the bacteria via this type III secretion system.  Some of these secreted proteins are components of the translocation apparatus that transfers other secreted proteins into the cytoplasm of the host cell.  One of the proteins that enters the host cell is called EspB [36] .  The precise function of EspB in the host cells is unknown, but EspB is required for translocation and many of the effects on host cells that have been observed.  Between the genes encoding the secretion apparatus and those encoding the secreted proteins lie two genes known as eae and tir, which encode intimin, an outer membrane adhesin required for virulence [9] , and Tir, its cognate receptor, respectively.  Tir is secreted by EPEC and inserted into the host cell membrane (Kenny et al. Cell 91:511-20, 1997) .
 
 
 
 

Diarrhea

    The mechanism(s) involved in EPEC diarrhea are still not known.  However, it is clear that EPEC require intimin [9] , EspB [45] and BFP (Bieber et al. Science 280:2114-8, 1998) to cause disease, as mutants unable to produce each of these factors are markedly attenuated compared to wild type when given to volunteers.  Recent studies have shown that EPEC induce a change in ion secretion in polarized intestinal epithelial cells and that this effect requires the type III secretion system and the Esp translocation apparatus proteins [37] .   This change in ion secretion may reflect electrolyte and fluid secretion in the intestine.  EPEC also cause a drop in tissue resistance that may correlate with loss of intestinal barrier function and passive loss of fluid into the intestinal lumen. The sole effector protein that is required for this loss of tissue resistance is EspF, which may therefore have a direct role in the process [49] .

Avoidance of Host Responses

    EPEC is one of the few known causes of persistant diarrhea.  The mechanisms that allow EPEC to avoid host defenses and persist in the intestine are unknown.  However, we have described a factor produced by EPEC that inhibits lymphocytes from proliferating and from producing cytokines [21] .  This factor is active on both human peripheral blood mononuclear cells and on mononuclear cells isolated from the lamina propria of the human intestine [27] .  Cloning of the gene encoding the factor, lifA, revealed a sequence predicted to encode a large protein related to the Clostridial cytotoxins [42] .  We call this protein lymphostatin.

     E. coli is the most common cause of urinary tract infections (UTI).  The strains that cause UTI differ from other E. coli in that they are more likely to possess certain factors, including adhesins and toxins, that are thought to play a role in the pathogenesis of UTI [BC3] .  However, few of these factors have been proven to be required for pathogenesis in animal models.  We used random (signature-tagged) mutagenesis and a murine model of ascending UTI to try to identify factors required for disease.  We found that type-1 fimbriae and capsule were particularly important [54] .  The entire sequence of the genome from a prototypic uropathogenic E. coli strain has now been completed and we participated in its annotation [55] .  This effort led to the identification of numerous new potential virulence factors, some of which we are actively investigating.