Sharon Ruschkowski

High‐frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein‐mediated invasion and cytoskeletal rearrangements

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90‐226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti‐M1 antibody, invasion by an M1− strain (90‐226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper‐like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal‐containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP‐1.

Inhibitors of cytoskeletal function and signal transduction to study bacterial invasion

Publisher Summary This chapter discusses the inhibitors of cytoskeletal function and signal transduction to study bacterial invasion. Cytochalasins are a class of drugs that inhibit actin polymerization, thereby blocking microfilament function; however, these drugs also have several other significant effects on cells. Uptake of most, if not all, bacteria into eukaryotic cells (including nonphagocytic cells such as those of epithelial origin) can be blocked by the use of cytochalasins. Cytochalasin D is considered to be the most specific and potent of the cytochalasins. To determine if this drug affects bacterial uptake, cultured cells are preincubated in 1μg/ml cytochalasin D (Sigma) for 30 min prior to infecting with bacteria and measuring invasion levels. It is becoming apparent that invasive bacteria have developed mechanisms with which they can exploit existing host signal transduction pathways to trigger cytoskeletal rearrangement and bacterial uptake. Many of these pathways use protein kinases to transmit signals, including kinases that phosphorylate serine and threonine residues, such as protein kinase C (PKC), and those that phosphorylate tyrosine residues, such as tyrosine protein kinases (TPKs). Agents are becoming available that inhibit these various enzymes, and thus they can be used to probe whether bacterial invasion involves a host kinase.

Tir Tyrosine Phosphorylation and Pedestal Formation Are Delayed in Enteropathogenic Escherichia coli sepZ::TnphoA Mutant 30-5-1(3)

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation. In this study, we show that 30-5-1(3), which has a transposon insertion within thesepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate. A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.

Isolation and characterization of the aadA aminoglycoside‐resistance gene from Salmonella choleraesuis

The streptomycin‐ and spectinomycin‐resistance gene of Salmonella choleraesuis was cloned and its nucleotide sequence determined. The gene is 789 bases long, encoding a protein of a predicted size of 29353 Da. The gene product inactivated streptomycin and spectinomycin by an adenylation modification. It is homologous (c. 40% total identity) to streptomycin adenylyltransferase, a 3′ (9)‐O‐nucleotidyltransferase (AAD(3′)(9)), which is encoded by the aad A gene in Escherichia coli, Agrobacterium tumefaciens, Kiebsiella pneumonia, and Serratia marcescens. The AadA protein of S. choleraesuis differs significantly from the other AadA proteins, indicating that it may have diverged from the other members of this family earlier in evolution. Southern hybridization analysis revealed that homologous aad A sequences were also present in other streptomycin‐resistant Salmonella species.

Enteropathogenic E. coli Exploitation of Host Epithelial Cells

Enteropathogenic E. coli (EPEC) is a leading cause of neonatal diarrhea worldwide. These organisms adhere to the intestinal cell surface, causing rearrangement in the epithelial cell surface and underlying cytoskeleton, resulting in a structure termed an attaching/effacing (A/E) lesion. A/E lesion formation is thought necessary for EPEC-mediated disease. EPEC secretes several proteins that trigger signal transduction, intimate adherence, and cytoskeletal rearrangements in epithelial cells. Additionally, it produces intimin, an outer membrane product that mediates intimate adherence. Together these various bacterial molecules contribute to the intimate relationship that is formed by EPEC with host epithelial cells which results in A/E lesion formation and diarrhea.