Multifunctional analysis of Chlamydia‐specific genes in a yeast expression system

Abstract

SummaryOur understanding of how obligate intracellular pathogens co-opt eukaryotic cellular functions has been limited by their intractability to genetic manipulation and by the abundance of pathogen-specific genes with no known functional homologues. In this report we describe a gene expression system to characterize proteins of unknown function from the obligate intracellular bacterial pathogen Chlamydia trachomatis . We have devised a homologous recombination-based cloning strategy to construct an ordered array of Saccharomyces cerevisiae strains expressing all Chlamydia -specific genes. These strains were screened to identify chlamydial proteins that impaired various yeast cellular functions or that displayed tropism towards eukaryotic organelles. In addition, to identify bacterial factors that are secreted into the host cell, recombinant chlamydial proteins were screened for reactivity towards antisera raised against vacuolar membranes purified from infected mammalian cells. We report the identification of 34 C. trachomatis proteins that impact yeast cellular functions or are tropic for a range of eukaryotic organelles including mitochondria, nucleus and cytoplasmic lipid droplets, and a new family of Chlamydia -specific proteins that are exported from the parasitopherous vacuole. The versatility of molecular manipulations and protein expression in yeast allows for the rapid construction of comprehensive protein expression arrays to explore the function of pathogen-specific gene products from microorganisms that are difficult to genetically manipulate, grow in culture or too dangerous for routine analysis in the laboratory.

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

“…Moreover, the other genes of this locus are arranged in operons (24) and predicted to encode important members of the T3SS apparatus, including the ATPase, chaperones, and membrane components (16). There is evidence that CT668, CT670, and CT671 are secreted proteins (47,51), and yet given their gene order they could represent mobile components of the T3SS apparatus. Indeed, CT670 contains an YscO-like domain raising the possibility that CT670 and CT671 correspond to the secreted Yersinia apparatus components YscO and YscP, respectively.…”

Identification of Coxiella burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation

“…Transformants were plated on uracil dropout media, supplemented with glucose, and incubated for 72 h at 30°C. Five colonies from each transformation were expanded, and toxicity was assessed by serially diluting and spotting onto the appropriate dropout media (27). Toxicity results were determined from at least three independent experiments.…”

Chlamydial Intracellular Survival Strategies

“…In addition to the Golgi, the inclusion interacts with other host-cell organelles, including (1) multivesicular bodies (MVBs) (Beatty 2006(Beatty , 2008Robertson et al 2009), which may also serve as a source of sphingolipids and cholesterol, (2) lipid droplets, which are translocated into the inclusion lumen following their capture by the chlamydial proteins Lda1 and/or Lda3 and which may serve as a source of neutral lipids (Kumar et al 2006;Cocchiaro et al 2008), (3) mitochondria (Matsumoto et al 1991;Derre et al 2007), and (4) lysosomes, which may be a source of essential amino acids derived from host-protein degradation (Ouellette et al 2011). Together, these interactions (summarized in Fig.…”

“…This includes gain-of-function approaches, such as expressing Chlamydia genes in heterologous systems to identify proteins that modulate host functions (Sisko et al 2006) or to identify proteins that can be secreted by surrogate type III secretion systems (Subtil et al 2001). Loss-offunction studies have also recently become possible with the implementation of targeting-induced local lesions in genomes (TILLING), an approach used extensively in plant and zebrafish to screen for mutants in genes of interest (reviewed in Stemple 2004).…”