Toxoplasma gondii Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells.

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Here, we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the Δtln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. IMPORTANCE After replicating within infected host cells, the single-celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study, we identify a parasite secretory protease that is necessary for efficient and timely egress, laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM+ (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.

Complete Annotated Genome Sequences of Three Campylobacter jejuni Strains Isolated from Naturally Colonized Farm-Raised Chickens.

Campylobacter jejuni is a leading cause of bacterially derived foodborne illness. Human illness is commonly associated with the handling and consumption of contaminated poultry products. Three C. jejuni strains were isolated from cecal contents of three different naturally colonized farm-raised chickens. The complete genomes of these three isolates are presented here.

Characterization and localization of the Campylobacter jejuni transformation system proteins CtsE, CtsP, and CtsX.

The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. Genes required for efficient transformation in C. jejuni include those similar to components of type II secretion systems found in many Gram-negative bacteria (R. S. Wiesner, D. R. Hendrixson, and V. J. DiRita, J Bacteriol 185:5408-5418, 2003, http://dx.doi.org/10.1128/JB.185.18.5408-5418.2003). Two of these, ctsE and ctsP, encode proteins annotated as putative nucleotide binding nucleoside triphosphatases (NTPases) or nucleoside triphosphate (NTP) binding proteins. Here we demonstrate that the nucleotide binding motifs of both proteins are essential for their function in transformation of C. jejuni. Localization experiments demonstrated that CtsE is a soluble protein while CtsP is membrane associated in C. jejuni. A bacterial two-hybrid screen identified an interaction between CtsP and CtsX, an integral membrane protein also required for transformation. Topological analysis of CtsX by the use of LacZ and PhoA fusions demonstrated it to be a bitopic, integral membrane protein with a cytoplasmic amino terminus and a periplasmic carboxyl terminus. Notwithstanding its interaction with membrane-localized CtsX, CtsP inherently associates with the membrane, requiring neither CtsX nor several other Cts proteins for this association.