Dissociation of Immune Responses from Pathogen Colonization Supports Pattern Recognition in C. elegans

Caenorhabditis elegans has been used for over a decade to characterize signaling cascades controlling innate immune responses. However, what initiates these responses in the worm has remained elusive. To gain a better understanding of the initiating events we delineated genome-wide immune responses to the bacterial pathogen Pseudomonas aeruginosa in worms heavily-colonized by the pathogen versus worms visibly not colonized. We found that infection responses in both groups were identical, suggesting that immune responses were not correlated with colonization and its associated damage. Quantitative RT-PCR measurements further showed that pathogen secreted factors were not able to induce an immune response, but exposure to a non-pathogenic Pseudomonas species was. These findings raise the possibility that the C.elegans immune response is initiated by recognition of microbe-associated molecular patterns. In the absence of orthologs of known pattern recognition receptors, C. elegans may rely on novel mechanisms, thus holding the potential to advance our understanding of evolutionarily conserved strategies for pathogen recognition

Sensing and Responding to Stress Stimuli in Caenorhabditis elegans: Implications for Aging and Immunity

Animals have evolved sophisticated mechanisms to perceive stress or even anticipate it before it has caused damage. Ideally, stress responses are controlled to ensure that they are specific and not excessive lest they cause collateral damage to the host. Here I describe my work in Caenorhabditis elegans examining the initiation and consequences of stress responses. In chapters 2 and 3, I examine the mechanisms of initiation of C.elegans immune responses to Pseudomonas aeruginosa, focusing on pathogen recognition. In chapters 4 and 5 I describe a project that was initiated within the realm of host-pathogen interactions, but evolved to focus on age as a context determining the outcome of both biotic and abiotic stress responses. Despite extensive knowledge of conserved signaling pathways in the C.elegans immune response, a question that has remained elusive is whether C.elegans can directly recognize pathogens, or if alternatively, they detect cellular damage caused by infection. I have shown that the immune response to P. aeruginosa can be dissociated from colonization and that potential damage from secreted molecules cannot induce a marker of the early immune response; in contrast, a non-pathogenic Pseudomonad can. This suggests that the response can be initiated at least in part by structural features of the bacteria. Furthermore, I have identified a family of genes in C.elegans encoding LysM domain proteins which are known to participate in recognition of microbial envelope components. Genetic and functional analyses suggest a potential role for family members in pathogen recognition in C. elegans. In the second half of this dissertation, I show that while the C.elegans JNK homolog KGB-1 is stress-protective during development, its activation in adults compromises stress resistance as well as general lifespan. I go on to show that this phenomenon is mediated in part by KGB-1's age-dependent antagonistic modulation of the conserved FOXO transcription factor, DAF-16. Genome-wide analysis of the KGB-1 transcriptome revealed that KGB-1 regulates several genes in an age-specific manner, and pointed to another conserved transcription factor, FOS-1 as a mediator of KGB-1's effects. Interestingly, the phenomenon of the same protein having opposite effects dependent on age is reminiscent of the Antagonistic Pleiotropy theory for the evolution of aging. Our results shed light on molecular mechanisms underlying this long-standing theory

Dissociation of Immune Responses from Pathogen Colonization Supports Pattern Recognition in C. elegans

Caenorhabditis elegans has been used for over a decade to characterize signaling cascades controlling innate immune responses. However, what initiates these responses in the worm has remained elusive. To gain a better understanding of the initiating events we delineated genome-wide immune responses to the bacterial pathogen Pseudomonas aeruginosa in worms heavily-colonized by the pathogen versus worms visibly not colonized. We found that infection responses in both groups were identical, suggesting that immune responses were not correlated with colonization and its associated damage. Quantitative RT-PCR measurements further showed that pathogen secreted factors were not able to induce an immune response, but exposure to a non-pathogenic Pseudomonas species was. These findings raise the possibility that the C.elegans immune response is initiated by recognition of microbe-associated molecular patterns. In the absence of orthologs of known pattern recognition receptors, C. elegans may rely on novel mechanisms, thus holding the potential to advance our understanding of evolutionarily conserved strategies for pathogen recognition

<i>C. elegans</i> immune responses to <i>Pseudomonas aeruginosa</i> are independent of colonization.

<p>A) 2-day old adult <i>C.elegans</i> exposed to GFP-expressing <i>P. aeruginosa</i> for 18 hrs show variability in colonization, allowing isolation of colonized and non-colonized worms. B) Gene expression profiles of <i>C. elegans</i> fed with <i>E. coli</i> or with GFP-expressing <i>P. aeruginosa</i> (colonized and non-colonized) for 18 hours; separation was achieved either by picking under a fluorescent stereoscope (1 experiment; grey bar), or using the COPAS^TM Worm Sorter (two independent experiments ; black bar). Shown are genes responding to the pathogen, as identified with a multi-class t-test analysis (10% false discovery rate).</p

<i>Pseudomonas</i> secreted factors are not sufficient to induce immune responses, while conserved cell-associated factors are.

<p>Gene expression measured by qRT-PCR, following exposure of young-adult <i>C. elegans</i> (at T₀) to <i>E. coli</i>, alone or with <i>P. aeruginosa</i> supernatant (A) or <i>P. aeruginosa</i> filtrate (B,C), or to the non-pathogenic <i>P. mendocina</i> (D).</p

Health professionals' overestimation of knowledge on snakebite management, a threat to the survival of snakebite victims-A cross-sectional study in Ghana.

BackgroundAccording to the World Health Organization, snakebites, a common occupational hazard in developing countries accounts for an annual loss of between 81,000 and 138, 000 lives following 5 million bites of which 2.7 million results in envenomation. Since snakebite-associated morbidity and mortality are more prevalent in agricultural economies such as Ghana, health professionals should be optimally knowledgeable on how to manage the incidence of snakebites. Lack of knowledge or overestimation of a professional's knowledge can affect heath delivery especially for emergencies such as snakebites. The three rurally situated Tongu districts in South-Eastern Ghana with agriculture as the major source of livelihood for their inhabitants, are prone to snakebites. This study, therefore, brings up the need to assess whether the health professionals in these districts are well-equipped knowledge-wise to handle such emergencies and whether they can rightly estimate their knowledge with regards to snakebite management.Methodology/principal findingsData was collected using a de novo semi-structured questionnaire administered through google form whose link was sent via WhatsApp to 186 health workers made up of nurses, midwives, physician assistants, medical doctors, pharmacists, and pharmacy technicians. This data was analyzed using Statistical Package for the Social Sciences (SPSS) Version 25. Association between variables was determined using the appropriate tools where necessary, using a confidence interval of 95% and significance assumed when p ≤ 0.05. This study found male health workers significantly more knowledgeable about snakebite management (11.53±5.67 vs 9.64±5.46; p = 0.022) but it was the females who overestimated their knowledge level (27.9% vs 24.1%). The medical doctors exhibited the best knowledge on snakebite management with the registered general nurses least knowledgeable. Although most professionals overestimated their knowledge, the registered general nurses were the worst at that (53.7%). Overall knowledge of health care professionals on snakebite management was below average [10.60±5.62/22 (48.2%)] but previous in-service training and involvement in the management of snakebite were associated with better knowledge. Respondents who had no previous training overestimated their knowledge level compared to those who had some post-qualification training on snakebite management (38.1% vs 7.5%). The greatest knowledge deficit of respondents was on the management of ASV associated adverse reactions.ConclusionHealth workers in rural Ghana overestimated their knowledge about snakebite management although their knowledge was low. Training schools, therefore, need to incorporate snakebite management in their curriculum and health authorities should also expose health workers to more in-service training on this neglected tropical disease

Transcriptional Profiling Identifies a Role for BrlA in the Response to Nitrogen Depletion and for StuA in the Regulation of Secondary Metabolite Clusters in Aspergillus fumigatus▿ ‡

Conidiation (asexual sporulation) is a key developmental process in filamentous fungi. We examined the gene regulatory roles of the Aspergillus fumigatus developmental transcription factors StuAp and BrlAp during conidiation. Conidiation was completely abrogated in an A. fumigatus ΔbrlA mutant and was severely impaired in a ΔstuA mutant. We determined the full genome conidiation transcriptomes of wild-type and ΔbrlA and ΔstuA mutant A. fumigatus and found that BrlAp and StuAp governed overlapping but distinct transcriptional programs. Six secondary metabolite biosynthetic clusters were found to be regulated by StuAp, while only one cluster exhibited BrlAp-dependent expression. The ΔbrlA mutant, but not the ΔstuA mutant, had impaired downregulation of genes encoding ribosomal proteins under nitrogen-limiting, but not carbon-limiting, conditions. Interestingly, inhibition of the target of rapamycin (TOR) pathway also caused downregulation of ribosomal protein genes in both the wild-type strain and the ΔbrlA mutant. Downregulation of these genes by TOR inhibition was associated with conidiation in the wild-type strain but not in the ΔbrlA mutant. Therefore, BrlAp-mediated repression of ribosomal protein gene expression is not downstream of the TOR pathway. Furthermore, inhibition of ribosomal protein gene expression is not sufficient to induce conidiation in the absence of BrlAp

The Developmental Intestinal Regulator ELT-2 Controls p38-Dependent Immune Responses in Adult C. elegans.

GATA transcription factors play critical roles in cellular differentiation and development. However, their roles in mature tissues are less understood. In C. elegans larvae, the transcription factor ELT-2 regulates terminal differentiation of the intestine. It is also expressed in the adult intestine, where it was suggested to maintain intestinal structure and function, and where it was additionally shown to contribute to infection resistance. To study the function of elt-2 in adults we characterized elt-2-dependent gene expression following its knock-down specifically in adults. Microarray analysis identified two ELT-2-regulated gene subsets: one, enriched for hydrolytic enzymes, pointed at regulation of constitutive digestive functions as a dominant role of adult elt-2; the second was enriched for immune genes that are induced in response to Pseudomonas aeruginosa infection. Focusing on the latter, we used genetic analyses coupled to survival assays and quantitative RT-PCR to interrogate the mechanism(s) through which elt-2 contributes to immunity. We show that elt-2 controls p38-dependent gene induction, cooperating with two p38-activated transcription factors, ATF-7 and SKN-1. This demonstrates a mechanism through which the constitutively nuclear elt-2 can impact induced responses, and play a dominant role in C. elegans immunity