Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host–pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N‐terminus and an immunogenic central region are not required for IpaC‐dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N‐terminus is essential, because IpaC mutants lacking the N‐terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane‐associating protein. Meanwhile, deletion of the C‐terminus eliminates the mutant protein's ability to establish protein–protein contacts with full‐length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact‐mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.

SummaryStreptococcus pyogenes , the aetiological agent of both respiratory and skin infections, produces numerous exotoxins to establish infection. This report identifies a new exotoxin produced by this organism, termed SpyA, for S. pyogenes ADP-ribosylating toxin. SpyA, MW 24.9, has amino acid identity with the ADP-riboslytransferases (ADPRTs) Staphylococcus aureus EDIN and Clostridium botulinum C3. Recombinant SpyA was able to hydrolyse b b b b -NAD + + + + , and this activity was dependent on a glutamate at position 187. SpyA has a putative biglutamate active site, and similar to most biglutamate ADPRTs, was able to ADP-ribosylate poly-L -arginine. SpyA modified numerous proteins in both CHO and HeLa cell lysates. Two-dimesional gel analysis and MALDI-TOF MS analysis of modified proteins indicated that vimentin, tropomyosin and actin, all cytoskeletal proteins, are targets. Expression of spyA in HeLa cells resulted in loss of actin microfilaments. We hypothesize that SpyA is produced by S. pyogenes to disrupt cytoskeletal structures and promote colonization of the host.

Rho-family GTPases are essential eukaryotic signaling molecules that regulate cellular physiology. Virulence factors from various pathogens alter GTPase signaling by functioning as GTPase activating factors (GAPs), guanine exchange factors (GEFs) or direct covalent modifiers. Bacterial virulence factors that sense rather than alter Rho-family GTPase signaling states have not been previously described. Here, we report that the translocated Salmonellae virulence factor SseJ binds to the GTP bound form of RhoA with resultant stimulation of its enzymatic activity that results in host cell membrane cholesterol esterification. Therefore, GTPase mediated downstream activation is not exclusive to eukaryotic proteins, and a bacterial protein has evolved to recognize the GTPase signaling state of RhoA to regulate its enzymatic activity as part of the host-pathogen interaction.

Invasion of enterocytes by Shigella flexneri requires the properly timed release of IpaB and IpaC at the host-pathogen interface; however, only IpaC has been found to possess quantifiable activities in vitro. We demonstrate here that when added to cultured cells, purified IpaC elicits cytoskeletal changes similar to those that occur during Shigella invasion. This IpaC effect may correlate with its ability to interact with model membranes at physiological pH and to promote entry by an ipaC mutant of S. flexneri.

Background: SpyA is a streptoccocal ADP-ribosyltransferase that modifies vimentin. Results: Vimentin is modified in the regulatory N-terminal head domain, and treatment with SpyA results in a defect in filamentation. Conclusion: Vimentin is a target substrate of SpyA, and ADP-ribosylation affects polymerization dynamics. Significance: Vimentin filaments are disrupted by a bacterial ADP-ribosyltransferase, SpyA, and ADP-ribosylation of vimentin regulates filament formation.