Reddy AB et al. (1984) J Helminthol "Microfilarial periodicity in Mastomys natalensis."

Subrahmanyam D, Reddy AB, Chandrashekar R, Rao UR

[J Helminthol, 1984]

The microfilarial level in the peripheral blood of Mastomys natalensis infected with the filarial parasites, Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae was monitored at two-hour intervals for 24 hours. The microfilariae of B. pahangi and B. malayi exhibited nocturnal and diurnal subperiodicity, respectively; no such periodicity was seen in L. carinii and D. viteae infections. The level of B. pahangi and D. viteae microfilariae in peripheral blood was significantly increased when the host was anaesthetized with diethylether or pentothal sodium. Ether-induced anaesthesia had no effect on the level of B. malayi microfilariae but pentothal was most effective. The peripheral blood count of L. carinii microfilariae tended to decrease in the anaesthetized animals but the reduction was not statistically significant.

Reddy J et al. (1989) International C. elegans Meeting "postreplication repair in C elegans."

Reddy J, Hartman PS

[International C. elegans Meeting, 1989]

Reddy KC et al. (2009) Science "A polymorphism in npr-1 is a behavioral determinant of pathogen susceptibility in C. elegans."

Kim DH, Andersen EC, KRUGLYAK L, Reddy KC

[Science, 2009]

The nematode Caenorhabditis elegans responds to pathogenic bacteria with conserved innate immune responses and pathogen avoidance behaviors. We investigated natural variation in C. elegans resistance to pathogen infection. With the use of quantitative genetic analysis, we determined that the pathogen susceptibility difference between the laboratory wild-type strain N2 and the wild isolate CB4856 is caused by a polymorphism in the npr-1 gene, which encodes a homolog of the mammalian neuropeptide Y receptor. We show that the mechanism of NPR-1-mediated pathogen resistance is through oxygen-dependent behavioral avoidance rather than direct regulation of innate immunity. For C. elegans, bacteria represent food but also a potential source of infection. Our data underscore the importance of behavioral responses to oxygen levels in finding an optimal balance between these potentially conflicting cues.

Kirthi Reddy et al. (2001) International C. elegans Meeting "Special handling for the X chromosome in meiosis"

Kirthi Reddy, Gillian Stanfield, Monica Colaiacovo, Stuart Kim, Valerie Reinke, Linwah Yip, Anne Villeneuve

[International C. elegans Meeting, 2001]

During prophase of meiosis I, homologous chromosomes must locate their partners, associate tightly along their lengths, and undergo recombination to ensure proper segregation at the first meiotic division. We are interested in the mechanisms that underly these processes, and have used a functional genomics approach to screen for components of the meiotic machinery in C. elegans. Microarray analysis has identified a set of genes whose expression is enriched in the C. elegans germline relative to somatic tissues (1). We used RNAi to inhibit the function of a subset of these genes that had expression profiles similar to those of known meiosis genes. For a high percentage of the genes analyzed, RNAi elicited germline phenotypes including meiotic defects, gonadogenesis defects, and abnormal chromatin in the mitotic zone of the germline. Among the genes for which RNAi caused a defect in meiotic chromosome segregation, one gene, T09E8.2, had a property that set it apart. Specifically, the relative frequencies of inviable embryos and males produced by affected animals suggested that depletion of T09E8.2 activity preferentially affected the X chromosome. It is not altogether surprising that the X chromosome could be more susceptible to perturbations that affect chromosome behavior, since the X has several properties that distinguish it from the autosomes. For example, each of the autosomes has a central gene-rich region in which recombination is suppressed, whereas both physical density of genes and recombination are more uniformly distributed on the X (2). Further, germline-enriched genes are notably absent from the X chromosome (1). X chromosomes also exhibit distinct chromatin properties during meiotic prophase (3,4). T09E8.2 therefore provides an entry point for understanding how general meiotic mechanisms intersect with special properties of sex chromosomes. The T09E8.2 RNAi phenotype was reminiscent of the meiotic defects we had previously observed in him-17 mutants. The meiotic segregation of both the autosomes and the X chromosome are affected in him-17 mutants, but the X chromosomes are much more strongly affected. There is a high frequency of achiasmate chromosomes in oocyte nuclei, presumably reflecting a defect in a mechanism that ensures crossing over. Interestingly, there is an alteration in both the frequency and distribution of crossovers along the X chromosome in him-17 mutants: recombination levels are elevated in a small region at the left end but are reduced along the remainder of the chromosome, suggesting a defect in associations between X chromosomes. Our genetic mapping data had localized him-17 to a 2 Mb interval containing T09E8.2. We therefore sequenced T09E8.2 in three independently isolated him-17 mutants and identified mutations in all three alleles. This demonstrates how the data from our RNAi screen can greatly aid in the cloning of genes identified by forward genetic screens by reducing the need for extensive mapping of mutants. We are currently investigating the role of HIM-17 during meiotic prophase. Initial analysis using fluorescence in situ hybridization indicates that in him-17 mutants, X chromosomes can initially pair but this paired state may not be stabilized. We are examining different chromosome regions for premature loss of pairing, and are exploring whether loss of pairing might reflect premature dissociation of synaptonemal complex proteins. 1. Reinke et al. (2000) Mol Cell 6(3):605-16. 2. Barnes et al. (1995) Genetics 141(1):159-79. 3. Goldstein and Slaton (1982) Chromosoma 84(4):585-97. 4. A. Dernburg and V. Reinke, personal communication.

Reddy, K. et al. (2017) International Worm Meeting "Probing organismal proteostasis through the response to intracellular infection."

Panek, J., Troemel, E., Dror, T., Reddy, K.

[International Worm Meeting, 2017]

Maintenance of protein homeostasis, or proteostasis, is critical for organismal health. Through analysis of the host transcriptional response to pathogen infection, we have identified a new pathway that appears to increase proteostasis capacity. We have previously reported that intracellular infection of C. elegans by either microsporidia or virus upregulates expression of several predicted components of a ubiquitin ligase complex (including the cullin cul-6) and many genes of unknown function (including pals-5) (Bakowski et al, PLoS Pathogens 2014). These genes are also induced by proteasome inhibition as well as by a prolonged heat stress at 30 deg . We have named this response the IPR (Intracellular Pathogen Response). Notably, the IPR is distinct from previously described pathways such as the heat shock response. Using a GFP reporter for the IPR gene pals-5, we isolated mutants that constitutively express IPR genes (including ubiquitin ligase components). These mutants are defective in the gene pals-22 (named for a domain implicated in the disease ALS), which functions as a repressor of the IPR. We found that pals-22 mutants have enhanced resistance to heat shock at 37 deg , and this phenotype is dependent on cul-6. We also examined whether pals-22 mutants have improved function in a polyglutamine (polyQ) aggregation assay. We found that pals-22 mutants have a greatly reduced number of polyQ aggregates in the intestine after osmotic shock compared to wild-type, and this phenotype is also dependent on cul-6. pals-22 mutants have no increased resistance to direct proteasome inhibition and no increased expression of proteasome subunits, indicating that the pals-22 phenotypes are not due to upregulation of proteasome function. Together these results suggest that upregulated expression of ubiquitin ligase components may increase the ability to target misfolded or toxic proteins for ubiquitylation and subsequent destruction in this mutant. The PALS-22 protein appears to be expressed broadly throughout the worm, including the intestine, hypodermis, and neurons. We are currently using tissue-specific rescue to identify the tissues in which pals-22 functions to affect stress resistance. We are also taking a biochemical approach to look for evidence of increased ubiquitylation in pals-22 mutants (see abstract by Panek et al).

Reddy KC et al. (2017) Curr Biol "An Intracellular Pathogen Response Pathway Promotes Proteostasis in C.elegans."

Lim ES, Dror T, Wang D, Sowa JN, Chen K, Troemel ER, Panek J, Reddy KC

[Curr Biol, 2017]

Maintenance of protein homeostasis, or proteostasis, is crucial for organismal health. Disruption of proteostasis can lead to the accumulation of protein aggregates, which are associated with aging and many human diseases such as Alzheimer's disease [1-3]. Through analysis of the C.elegans host response to intracellular infection, we describe here a novel response pathway that enhances proteostasis capacity and appears to act in parallel towell-studied proteostasis pathways. These findings are based on analysis of the transcriptional response to infection by the intracellular pathogen Nematocida parisii [4]. The response to N.parisii is strikingly similar to the response to infection bythe Orsay virus, another natural intracellular pathogen of C.elegans, and is distinct from responses to extracellular pathogen infection [4-6]. We have therefore named this common transcriptional response the intracellular pathogen response (IPR), and it includes upregulation of several predicted ubiquitin ligase complex components such as the cullin cul-6. Through a forward genetic screen we found pals-22, a gene of previously unknown function, to be a repressor of the cul-6/cullin gene and other IPR gene expression. Interestingly, pals-22 mutants have increased thermotolerance and reduced levels of stress-induced polyglutamine aggregates, likely due to upregulated IPR gene expression. We found the enhanced stress resistance of pals-22 mutants to be dependent on cul-6, suggesting that pals-22 mutants have increased activity of a CUL-6/cullin-containing ubiquitin ligase complex. pals-22 mutant phenotypes appear independent of the well-studied heat shock and insulin signaling pathways, indicating that the IPR is a distinct pathway that protects animals from proteotoxic stress.

Reddy KC et al. (2016) Cell Rep "The C.elegans CCAAT-Enhancer-Binding Protein Gamma Is Required for Surveillance Immunity."

Reddy KC, Troemel ER, Haynes CM, Nargund AM, Dunbar TL

[Cell Rep, 2016]

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C.elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C.elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P.aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P.aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses.

Bhaskar Reddy Gavinolla et al. (2007) International Worm Meeting "Interactions between the microtubule severing protein MEI-2 and spindle checkpoints."

Bhaskar Reddy Gavinolla, Paul E. Mains

[International Worm Meeting, 2007]

The spindle checkpoint protein, SAN-1/MDF-3 is expressed on mitotic centromeres. During anoxia, mutants fail to arrest the cell cycle, leading to chromosome mis-segregation and reduced viability (Nystul et. al 2003). Checkpoint proteins arrest the cell cycle by inhibiting the Anaphase Promoting Complex (APC) and san-1/mdf-3 mutants suppress APC mutants (Stein et al. 2007). We have uncovered a possible link between the microtubule severing complex MEI-1/MEI-2 and the meiotic spindle checkpoint. Like SAN-1/MDF-3, MEI-1 and MEI-2 localize to chromatin and genetically interact with APC mutants. Furthermore, yeast two hybrid data (Li et al. 2004) shows that MEI-2 and SAN-1 interact physically. Both the similar expression patterns and the yeast two-hybrid binding suggested possible genetic interaction between them, so we made double mutants of san-1 and mei-2. This resulted in increased lethality, low brood sizes and spontaneous males, indications of meiotic failure. While enhancement of mei-2 spindle formation defects might be expected by the presence of a compromised spindle checkpoint, the physical interaction and colocalization might indicate a more specific role for MEI-1/MEI-2 in monitoring spindle quality.

Kirthi C Reddy et al. (2003) International Worm Meeting "HIM-17, a novel chromatin-associated protein required for initiation of meiotic recombination"

Kirthi C Reddy, Anne M Villeneuve

[International Worm Meeting, 2003]

During prophase of meiosis I, homologous chromosomes must locate their partners and undergo crossover recombination to ensure correct segregation at the first meiotic division. We have used a combination of classical genetics and functional genomics to identify HIM-17, a novel protein that is required for formation of crossovers. We have obtained five mutant alleles of him-17, including one that appears to be a null allele. All five alleles result in defects in meiotic chromosome segregation, as evidenced by production of high frequencies of both males and inviable embryos (indicative of X chromosome and autosomal missegregation, respectively). We have strong evidence that HIM-17 is required for the formation of the double-strand DNA breaks (DSBs) that initiate meiotic recombination. First, recombination intermediates downstream of DSB formation are not detected in him-17 mutants. Second, depletion of RAD-51, a protein required for proper repair of DSBs, does not lead to altered chromosome morphology in the him-17 null mutant. Finally, ionizing radiation (which artificially generates DSBs) can bypass the defect in recombination seen in him-17, producing functional chiasmata. This response to ionizing radiation is identical to that seen in worms lacking SPO-11, a protein that is believed to generate DSBs (1). We therefore conclude that SPO-11-induced breaks are not being formed in him-17 mutants. Additional data suggest that HIM-17 promotes DSB formation by modifying chromatin to allow DSBs. We have generated a HIM-17::GFP translational fusion that fully rescues the null allele, and have shown that this fusion protein localizes to chromatin in germline nuclei. In addition, while all six pairs of chromosomes require HIM-17 for formation of crossovers, the X chromosomes are much more affected by a partial loss of HIM-17 function. The X chromosomes have different chromatin properties than the autosomes in wild-type meiosis (2), which could explain why the X relies more on HIM-17 function. Finally, we find that antibodies against histone H3 methyl-Lys9 exhibit an altered staining pattern in him-17 mutant germlines, indicative of aberrant chromatin organization. We are currently doing experiments to further investigate interactions between chromatin and recombination proteins in him-17 mutants. 1. Dernburg et al. Cell 94: 387-398. 2. Kelly et al. Development 129: 479-492.

Reddy KC et al. (2019) PLoS Pathog "Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans."

Barkoulas M, Desjardins CA, Dror T, Underwood RS, Troemel ER, Cuomo CA, Osman GA, Elder CR, Reddy KC

[PLoS Pathog, 2019]

Immune genes are under intense pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of "Intracellular Pathogen Response" or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans.