Showing papers by "B. Brett Finlay published in 1993"

Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells

Abstract: Salmonella species invade and replicate within epithelial cells in membrane-bound vacuoles. In this report we show that upon infection of HeLa epithelial cells, Salmonella typhimurium residues in vacuoles that contain lysosomal membrane glycoproteins (lgps). Four to six hours after invasion, intracellular bacteria induce the formation of stable filamentous structures containing lgps that are connected to the bacteria-containing vacuoles. Formation of these lgp-rich structures requires viable intracellular bacteria and is blocked by inhibitors of vacuolar acidification. These structures are not present in uninfected cells or in cells infected with another invasive bacteria, Yersinia enterocolitica. Tracers added to the extracellular medium are not delivered to the Salmonella-induced filaments, suggesting that these structures are different from previously described tubular lysosomes. Initiation of intracellular bacterial replication correlates with formation of these lgp-containing filaments. Certain avirulent Salmonella mutants that are defective for intracellular replication fail to induce formation of these structures. These observations suggest that Salmonella-induced filaments containing lgps are linked to intracellular bacterial replication.

Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.

Abstract: The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined. We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells. This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin. Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably affected in the epithelial cells, nor was the distribution of ZO-1, a tight junction protein. Despite the decrease in transepithelial electrical resistance, EPEC did not cause an increase in [3H]inulin penetration across MDCK monolayers. Unlike in the parental strain, mutations in any loci involved in adherence or formation of attaching and effacing lesions were unable to cause a decrease in transepithelial resistance. These data indicate that EPEC causes a decrease in transepithelial electrical resistance by disrupting a transcellular (intracellular) pathway rather than by disrupting intercellular tight junctions (paracellular) and that these disruptions occur only when attaching and effacing lesions are formed.

Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells

Abstract: Our laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host-adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147-amino-acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of approximately 16,000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.

Role of acid tolerance response genes in Salmonella typhimurium virulence.

Abstract: The atp and fur genes are involved in the acid tolerance response of Salmonella typhimurium. An atp::Tn10 mutant was avirulent in the mouse typhoid model when assayed by oral and intraperitoneal routes. However, a fur mutant was completely virulent by the intraperitoneal route. No relevant differences in intracellular survival or invasion rates were observed for the two mutants in macrophages and epithelial cells. These data indicate that separate acid tolerance response genes may have different roles in S. typhimurium virulence.

Intracellular replication of Salmonella within epithelial cells is associated with filamentous structures containing lysosomal membrane glycoproteins.

Abstract: We have examined the targeting of S. typhimurium-containing vacuoles to lysosomes after invasion of cultured HeLa epithelial cells. Our results show that intracellular bacteria colocalize with vacuoles containing lysosomal membrane glycoproteins (LGPs). Both human LGPs, hlamp-1 and hlamp-2, are present in S. typhimurium-containing vacuoles from approximately 2 h postinfection. At later times (4-6 h), long and stable filamentous structures with lysosomal markers appear connected to bacteria-containing vacuoles in infected cells. Viable intracellular bacteria are required for the formation of these structures, which are not detected in uninfected cells or in HeLa epithelial cells infected with another invasive bacteria, Yersinia enterocolitica. Kinetics analysis showed a strict correlation between the appearance of these LGP-rich filaments and the initiation of intracellular bacterial replication. Moreover, these structures are absent in epithelial cells infected with certain S. typhimurium intracellular replication-defective mutants. Additional data confirmed that an intact microtubule network and intravacuolar acidic pH are required to induce the formation of LGP-containing filamentous structures and that these structures are morphologically and functionally different from previously described tubular lysosomes.

Intracellular Trafficking of Salmonella Typhimurium within HeLa Epithelial Cells

Abstract: Salmonella typhimurium is an intracellular pathogen which penetrates (invades) and multiplies within epithelial cells (Finlay and Falkow, 1988, 1989). Unlike some intracellular pathogens, S. typhimurium remains within a vacuole (Finlay and Falkow, 1988, 1989). Following internalization this vacuole migrates to the perinuclear region, and after a lag period of 2–3 h, bacteria initiate intracellular multiplication (Finlay and Falkow, 1989). We are currently characterizing the intracellular trafficking of bacteria-containing vacuoles.