The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion

Abstract

Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.

Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

“…Deletion of etgA from the EPEC homolog Citrobacter rodentium, a mouse bacterial pathogen similar to EPEC, impedes T3S and attenuates virulence (29). Accordingly, deletion of etgA from EPEC decreases T3SS assembly and reduces T3SS-dependent hemolysis of erythrocytes (26). However, it has been reported that disruption of the Salmonella ipgF gene had no effect on HeLa cell invasion, although this may not be reflective of the situation in the naturally targeted epithelial cells (30).…”

“…The ⌬etgA mutant of both C. rodentium and EPEC displayed a reduced type III secretion phenotype. The Citrobacter ⌬etgA mutant exhibited a more than 90% reduction in type III secretion when compared with the wildtype strain (29), whereas the EPEC ⌬etgA mutant only had 50% attenuation of type III secretion, possibly because of functional redundancy with a housekeeping PG-lytic enzyme (data not shown; (26)). Because greater reduction in type III secretion was useful for testing the effect of EtgA catalytic mutants, the Citrobacter ⌬etgA mutant was used for the following experiments.…”

“…EtgA was classified as a putative family 1A lytic transglycosylase because of the presence of conserved sequence motifs (11). The N terminus of EtgA has a signal peptide that targets it for transport to the periplasm by the Sec-dependent general secretory pathway (26). An early study that used a yeast two-hybrid assay to survey interactions between LEE encoded proteins suggested that EtgA binds the inner rod subunit, EscI (27).…”

“…An early study that used a yeast two-hybrid assay to survey interactions between LEE encoded proteins suggested that EtgA binds the inner rod subunit, EscI (27). Peptidoglycan degrading activity of EtgA, as well as T3SS PG-lytic enzyme homologs from Salmonella and Shigella (IagB and IpgF, 41 and 35% sequence identity, respectively), was shown by zymogram, and mutation of a conserved glutamate (Glu-42 in EPEC) abrogates activity (26,28). Deletion of etgA from the EPEC homolog Citrobacter rodentium, a mouse bacterial pathogen similar to EPEC, impedes T3S and attenuates virulence (29).…”

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…To date, the contribution of predicted LTs to T3S and/or pathogenicity has been studied in both animal-and plant-pathogenic bacteria (75,192,412,413,624,625,628) (summarized in Table 3). Notably, it was observed that single LTs do not contribute significantly to T3S and virulence, presumably due to functional redundancies.…”