SummaryBordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica , the screen identified the first TTSS chaperone-effector locus, btcA-bteA , and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica , it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis . Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-β-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.

Tyrosine phosphorylation of phospholipase C␥2 (PLC␥2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of nonreceptor tyrosine kinases can phosphorylate PLC␥ in vitro, the specific kinase(s) controlling BCR-dependent PLC␥ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLC␥2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLC␥2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLC␥2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btkdeficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr 753 and Tyr 759 . Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLC␥2 carboxyl-terminal sites, Tyr 1197 and Tyr 1217 , was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLC␥2 SH2-SH3 linker.Signals generated by the pre-B and mature B cell receptors are essential for B cell development, activation, and maintenance of mature B cell populations (1). Engagement of the BCR 1 initiates the formation of a lipid-raft associated signaling complex, or "signalosome," containing tyrosine and serine/threonine kinases, adapter molecules, and lipid hydrolases including phospholipase C␥ isoforms. Together, these events promote a series of downstream signals including a sustained increase in intracellular calcium concentrations. PLC␥ is essential for antigen receptor-mediated calcium mobilization (2-4). Activated PLC␥ hydrolyzes its substrate, phosphatidylinositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (5, 6). IP 3 acts to open intracellular calcium stores promoting an initial, transient rise in intracellular calcium. Depletion of intracellular calcium stores triggers the opening of plasma membrane store-operated calcium channels (7), resulting in an influx of extracellular calcium and a secondary, sustained calcium signal. The amplitude and duration of this sustained calcium signal is a key determinant of the specific transcription program initiated in response to antigen receptor engagement (8 -10).The most abundantly expressed PLC␥ isoform in B lineage cells is PLC␥2. Chicken B lymphoma cells lacking PLC␥2 are unable to generate IP 3 in response to BCR engagement, resulting in the abrogation of receptor-mediated calcium mobilization (11). Similarly, mice deficient in...

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