No abstract

Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. Here we identify genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila melanogaster challenged with Salmonella enterica serovartyphimurium (S. typhimurium). We demonstrate that wild-type S. typhimurium causes a lethal systemic infection when injected into the hemocoel of D. melanogaster. Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. We propose a model in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, we show that mutations in the fly gene eiger, a TNF homolog, delay the lethality of Salmonella infection. These results suggest that S. typhimurium-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides us with a new model to study shock-like biology in a genetically manipulable host. In addition, it allows us to study the difference in pathways followed by a microbe when producing an acute or persistent infection.

We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger.

Chemokines inhibit entry of HIV into CD4؉ T cells more effectively than into macrophages or transfected adherent cells. Here, we tested whether chemokine receptor internalization could account for cell type differences in the effectiveness of chemokines. Infection of CEM T cells expressing stably transduced wild-type CCR5 was much more readily inhibited by chemokine than were transduced HOS cells. This response correlated with the efficiency of CCR5 internalization. A mutated CCR5, termed M7-CCR5, in which the Ser/Thr phosphorylation sites in the cytoplasmic tail were changed to Ala, did not internalize in response to MIP-1␣. M7-CCR5 was expressed at slightly higher levels than wild-type on stably transduced cell lines and was somewhat more potent as an HIV-1 coreceptor. The mutated receptor mobilized intracellular Ca 2؉ in response to chemokine to a level 4-fold higher than did the wild type CCR5. Unexpectedly, the receptor was desensitized as efficiently as wild type, suggesting that desensitization does not require cytoplasmic tail phosphorylation. Entry of R5 HIV-1 reporter virus into cells stably expressing M7-CCR5 was largely resistant to blocking by MIP-1␣. As much as 80% of entry inhibition was attributed to receptor internalization. Aminooxypentane (AOP)-MIP-1␣ was able to induce a low level of M7-CCR5 internalization in HOS and to weakly inhibit HIV-1 entry. Introduction of dominant negative dynamin into HOS cells reduced the ability of chemokine to inhibit infection. The inefficiency of internalization of chemokine receptors in some cell types could allow virus to replicate in vivo in the presence of endogenous chemokine. Last, M7-CCR5 is a useful tool for discriminating coreceptor internalization from binding site masking in the evaluation of small molecule inhibitors of HIV-1 entry.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.

Using PCR–denaturing gradient gel electrophoresis, we examined 49 fecal samples from healthy volunteers and 128 diarrhea specimens to assess the distribution of Epsilonproteobacteria that might be routinely overlooked. Our results suggest that certain taxa that are not routinely examined for could account for a proportion of diarrhea of previously unknown etiology.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes diarrheal disease in humans and is of public health concern because of its ability to cause outbreaks and severe disease such as hemorrhagic colitis or hemolytic-uremic syndrome. More than 400 serotypes of STEC have been implicated in outbreaks and sporadic human disease. The aim of this study was to develop a PCR binary typing (P-BIT) system that could be used to aid in risk assessment and epidemiological studies of STEC by using gene targets that would represent a broad range of STEC virulence genes. We investigated the distribution of 41 gene targets in 75 O157 and non-O157 STEC isolates and found that P-BIT provided 100% typeability for isolates, gave a diversity index of 97.33% (compared with 99.28% for XbaI pulsed-field gel electrophoresis [PFGE] typing), and produced 100% discrimination for non-O157 STEC isolates. We identified 24 gene targets that conferred the same level of discrimination and produced the same cluster dendrogram as the 41 gene targets initially examined. P-BIT clustering identified O157 from non-O157 isolates and identified seropathotypes associated with outbreaks and severe disease. Numerical analysis of the P-BIT data identified several genes associated with human or nonhuman sources as well as high-risk seropathotypes. We conclude that P-BIT is a useful approach for subtyping, offering the advantage of speed, low cost, and potential for strain risk assessment that can be used in tandem with current molecular typing schema for STEC.