David William Holden

FUNCTIONAL ANALYSIS OF SSAJ AND THE SSAK/U OPERON, 13 GENES ENCODING COMPONENTS OF THE TYPE III SECRETION APPARATUS OF SALMONELLA PATHOGENICITY ISLAND 2

We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10u2003kb. ssaJ lies upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild‐type strain, but none are defective in replication within macrophage‐like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild‐type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

The Aspergillus fumigatus chsC and chsG genes encode Class III chitin synthases with different functions

Two genes, designated chsC and chsG were isolated from DNA libraries of the opportunistic fungal pathogen, Aspergillus fumigatus. The genes were characterized with respect to their nucleotide sequences and mutant phenotypes. The complete deduced amino acid sequences of chsC and chsG show that the products of both genes are Class III zymogen‐type enzymes. A mutant strain constructed by disruption of chsC is phenotypically indistinguishable from the wild‐type strain, but chsG− and chsC−u2003chsG− strains have reduced colony radial growth rate and chitin synthase activity, conidiate poorly and produce highly branched hyphae. Despite these defects, the double‐mutant strain retained the ability to cause pulmonary disease in neutropenic mice. However, in comparison to the wild‐type strain, there was a decrease in mortality and delay in the onset of illness in mice inoculated with the double‐mutant strain, which was associated with smaller and more highly branched fungal colonies in lung tissue.

A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus

Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus λ genomic DNA library. The polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the ‘zymogen type’ chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

Molecular genetics of Aspergillus pathogenicity.

Aspergillus fumigatus is the most frequent cause of Invasive Pulmonary Aspergillosis (IPA), a life-threatening disease of immunosuppressed patients. In addition to a number of general physiological attributes of this fungus, it has been suggested that extracellular elastase and toxins might facilitate its growth in lung tissue. We have investigated the roles of two extracellular proteins, an alkaline protease with elastase activity (AFAlp), and the ribotoxin restrictocin in murine models of IPA. Gene disruption was used to create stable null mutant strains of the fungus lacking one or other protein, and their virulence and histopathological features were compared with an isogenic parental strain in steroid-treated and neutropenic mice. We have been unable to demonstrate any significant differences between the three strains, which shows that, considered independently, these proteins are not important virulence determinants. We are also interested in identifying fungal-specific gene products involved in general metabolism and which are required for growth in the lung, because these could represent new targets for antifungal drugs. For this work a model of murine IPA involvingAspergillus nidulans was established, to take advantage of the many well characterised mutations affecting metabolic pathways. Pathogenicity tests with strains carrying one of two auxotrophic mutations,lysA2 andpabaA1, have shown while lysine biosynthesis is not essential for the fungus to cause pulmonary disease, biosynthesis ofp-aminobenzoic acid is essential. We are now in the process of cloning theA. fumigatus pabaA homologue to determine its function and whether this gene is required for growth of the clinically important species in the lung.

Detection of gene-disruption events in aspergillus transformants by polymerase chain reaction direct from conidiospores

We describe a rapid method for the identification of gene disruption events after DNA-mediated transformation of Aspergilus fumigatus. This involves a polymerase chain reaction in which the target DNA is added in the form of intact conidiospores. Using one primer specific to the plasmid DNA and a second primer specific to the target gene on the chromosome, it is possible to identify gene disruption events among the more common ectopic integrations approximately 4 h after sporulating transformants appear on selective medium.

7.3 Signature Tagged Mutagenesis

Publisher Summary This chapter provides an overview of signature tagged mutagenesis (STM). STM combines the strength of mutational analysis with the ability to follow the fate of a large number of different mutants within a single animal. In its original form, a transposon was used for random insertional mutagenesis of the genome of Salmonella typhimurium , but there is no reason the technique could not be applied to other pathogens, so long as they are haploid, can undergo insertional mutagenesis, and infect their experimental host as a mixed population. In STM, each transposon contains a different DNA sequence tag, which allows mutants to be differentiated from each other. The tags comprise 40 bp variable central regions flanked by invariant “arms” of 20 bp, which allow the amplification and labeling of the central portions by the polymerase chain reaction (PCR). Mutants are assembled into 96-well microtiter dishes, which are used to prepare replica DNA colony blots. The mutants are then mixed to form the inoculum or “input” pool before inoculation into an animal. After infection is established, cells of the pathogen are isolated from the animal and pooled to form the “recovered pool.” Tags in the recovered pool and tags in the input pool are separately amplified, labeled, and used to probe DNA colony blots of the inoculum. The chapter further describes virulence gene cloning and DNA sequencing.