Ranjiv Khush

The Drosophila caspase Dredd is required to resist gram-negative bacterial infection.

The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide‐encoding genes through distinct signaling cascades. Fungal infection activates NF‐κB‐like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. We have isolated loss‐of‐function mutations in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by Gram‐negative bacteria. Our results demonstrate that Dredd regulates antibacterial peptide gene expression, and we propose that Dredd, Immune Deficiency and the P105‐like rel protein Relish define a pathway that is required to resist Gram‐negative bacterial infections.

Genes that fight infection: what the Drosophila genome says about animal immunity.

From deciphering the principles of heredity to identifying the genes that control development, the fruit fly Drosophila melanogaster is being used to deconstruct an increasing number of biological processes. Genetic studies of Drosophila responses to microbial infection have identified regulators of innate immunity that are functionally conserved in mammals. These recent findings highlight the ancient origins of animal immune responses and demonstrate the potential of Drosophila for dissecting host-pathogen interactions. The sequencing of the Drosophila genome both enhances genetic approaches and provides new clues for the identification of key components of innate immunity. This article summarizes how information gained from genomic analysis contributes to our understanding of how animals cope with infectious disease.

Drosophila immunity: two paths to NF-κB

Recent studies of Drosophila immune responses have defined the immune deficiency (IMD) signaling pathway that mediates defense against Gram-negative bacterial infection. Like the Toll pathway, the IMD pathway regulates antimicrobial peptide gene expression via a Rel/nuclear factor (NF)-kappaB-like transcription factor. However, the two pathways do not appear to share any intermediate components. Maintaining distinct immune response pathways might be one mechanism by which flies mount adapted immune responses.

Secreted Bacterial Effectors and Host-Produced Eiger/TNF Drive Death in a Salmonella-Infected Fruit Fly

Death by infection is often as much due to the hosts 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.

A ubiquitin-proteasome pathway represses the Drosophila immune deficiency signaling cascade

BACKGROUND The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-kappaB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-kappaB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-kappaB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. RESULTS In a screen for factors that negatively regulate the IMD pathway, we isolated two partial loss-of-function mutations in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and dCullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. CONCLUSIONS In contrast to other NF-kappaB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-kappaB downregulation in Drosophila.

Causal inference methods to study nonrandomized, preexisting development interventions

Empirical measurement of interventions to address significant global health and development problems is necessary to ensure that resources are applied appropriately. Such intervention programs are often deployed at the group or community level. The gold standard design to measure the effectiveness of community-level interventions is the community-randomized trial, but the conditions of these trials often make it difficult to assess their external validity and sustainability. The sheer number of community interventions, relative to randomized studies, speaks to a need for rigorous observational methods to measure their impact. In this article, we use the potential outcomes model for causal inference to motivate a matched cohort design to study the impact and sustainability of nonrandomized, preexisting interventions. We illustrate the method using a sanitation mobilization, water supply, and hygiene intervention in rural India. In a matched sample of 25 villages, we enrolled 1,284 children <5 y old and measured outcomes over 12 mo. Although we found a 33 percentage point difference in new toilet construction [95% confidence interval (CI) = 28%, 39%], we found no impacts on height-for-age Z scores (adjusted difference = 0.01, 95% CI = −0.15, 0.19) or diarrhea (adjusted longitudinal prevalence difference = 0.003, 95% CI = −0.001, 0.008) among children <5 y old. This study demonstrates that matched cohort designs can estimate impacts from nonrandomized, preexisting interventions that are used widely in development efforts. Interpreting the impacts as causal, however, requires stronger assumptions than prospective, randomized studies.

H2S as an Indicator of Water Supply Vulnerability and Health Risk in Low-Resource Settings: A Prospective Cohort Study

In this large-scale longitudinal study conducted in rural Southern India, we compared a presence/absence hydrogen sulfide (H2S) test with quantitative assays for total coliforms and Escherichia coli as measures of water quality, health risk, and water supply vulnerability to microbial contamination. None of the three indicators showed a significant association with child diarrhea. The presence of H2S in a water sample was associated with higher levels of total coliform species that may have included E. coli but that were not restricted to E. coli. In addition, we observed a strong relationship between the percent positive H2S test results and total coliform levels among water source samples (R(2) = 0.87). The consistent relationships between H2S and total coliform levels indicate that presence/absence of H2S tests provide a cost-effective option for assessing both the vulnerability of water supplies to microbial contamination and the results of water quality management and risk mitigation efforts.

Reactivity in Rapidly Collected Hygiene and Toilet Spot Check Measurements: A Cautionary Note for Longitudinal Studies

Discreet collection of spot check observations to measure household hygiene conditions is a common measurement technique in epidemiologic studies of hygiene in low-income countries. The objective of this study was to determine whether the collection of spot check observations in longitudinal studies could itself induce reactivity (i.e., change participant behavior). We analyzed data from a 12-month prospective cohort study in rural Tamil Nadu, India that was conducted in the absence of any hygiene or toilet promotion activities. Our data included hygiene and toilet spot checks from 10,427 household visits. We found substantial evidence of participant reactivity to spot check observations of hygiene practices that were easy to modify on short notice. For example, soap observed at the households primary handwashing location increased from 49% at enrollment to 81% by the fourth visit and remained at or above 77% for the remainder of the study.

To What Extent is Drinking Water Tested in Sub-Saharan Africa? A Comparative Analysis of Regulated Water Quality Monitoring

Water quality information is important for guiding water safety management and preventing water-related diseases. To assess the current status of regulated water quality monitoring in sub-Saharan Africa, we evaluated testing programs for fecal contamination in 72 institutions (water suppliers and public health agencies) across 10 countries. Data were collected through written surveys, in-person interviews, and analysis of microbial water quality testing levels. Though most institutions did not achieve the testing levels specified by applicable standards or World Health Organization (WHO) Guidelines, 85% of institutions had conducted some microbial water testing in the previous year. Institutions were more likely to meet testing targets if they were suppliers (as compared to surveillance agencies), served larger populations, operated in urban settings, and had higher water quality budgets (all p < 0.05). Our results indicate that smaller water providers and rural public health offices will require greater attention and additional resources to achieve regulatory compliance for water quality monitoring in sub-Saharan Africa. The cost-effectiveness of water quality monitoring should be improved by the application of risk-based water management approaches. Efforts to strengthen monitoring capacity should pay greater attention to program sustainability and institutional commitment to water safety.

Can Sanitary Surveys Replace Water Quality Testing? Evidence from Kisii, Kenya

Information about the quality of rural drinking water sources can be used to manage their safety and mitigate risks to health. Sanitary surveys, which are observational checklists to assess hazards present at water sources, are simpler to conduct than microbial tests. We assessed whether sanitary survey results were associated with measured indicator bacteria levels in rural drinking water sources in Kisii Central, Kenya. Overall, thermotolerant coliform (TTC) levels were high: all of the samples from the 20 tested dug wells, almost all (95%) of the samples from the 25 tested springs, and 61% of the samples from the 16 tested rainwater harvesting systems were contaminated with TTC. There were no significant associations between TTC levels and overall sanitary survey scores or their individual components. Contamination by TTC was associated with source type (dug wells and springs were more contaminated than rainwater systems). While sanitary surveys cannot be substituted for microbial water quality results in this context, they could be used to identify potential hazards and contribute to a comprehensive risk management approach.