BackgroundChlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates.ResultsWe first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells.ConclusionsUsing Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA 241) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin384) or the shorter (Lysin241) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various Gram-positive bacteria and mycobacteria.

Despite the recent advances achieved in food industries to fulfil the growing consumer demand for high quality and food safety, microbial contamination remains a serious issue. This study aimed to incorporate ϕIBB-PF7A bacteriophage (phage) onto sodium alginate-based films crosslinked with calcium chloride, to prevent poultry spoilage caused by Pseudomonas fluorescens. Films were prepared by casting and characterized in terms of phage loading, distribution, stability, release profile and antimicrobial performance. Results showed that phages were successfully incorporated as evidenced by their viability and homogeneous distribution within the films as assessed by microscopy. A decrease in phage viability was only detected after 8 weeks when stored under refrigerated conditions. Antimicrobial activity demonstrated that incorporated phages significantly impaired P. fluorescens growth. Films' antimicrobial efficacy was further demonstrated on chicken breast fillets artificially inoculated, decreasing 2Log P. fluorescens viable cell counts in the first two days and reductions were maintained up to 5 days of exposure (1 Log). These results highlight that phage incorporation onto sodium-alginate-based films constitutes a simple approach of preserving the antimicrobial activity of phages in a dried and insoluble format, that can further be applied in food industry for the prevention of microbial spoilage.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.