We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.RecQ helicases perform essential roles in maintaining genome stability (5). Mammals have five RecQ homologs: RecQL1, BLM/ RecQL2, WRN/RecQL3, RecQL4, and RecQL5 (6, 12). Mutations in BLM, WRN, and RecQL4 give rise to the genomic instability disorders Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome, respectively. These disorders are characterized by cancer predisposition, chromosomal instability, and cellular hypersensitivity to DNA-damaging agents. Although RecQL5 has not been associated with any human disease, RecQL5 Ϫ/Ϫ mice exhibit an increased incidence of cancer, a phenotype common to all RecQ helicase syndromes (5,16,20).RecQL5 may play a role in the stabilization and/or restart of stalled replication forks. This was suggested by findings that mouse RecQL5 Ϫ/Ϫ embryonic stem (ES) cells and primary embryonic fibroblasts are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor that blocks DNA replication (18,19). In addition, RecQL5 may suppress homologous recombination (HR) and/or crossover events, as evidenced by the observation that mouse RecQL5 Ϫ/Ϫ cells display an elevated frequency of sister chromatid exchange (SCE) (18,19). The roles of RecQL5 in the suppression of SCE can be replaced functionally by BLM in chicken DT40 cells, because the deletion of RecQL5 in normal DT40 cells does not lead to an elevated SCE frequency, whereas the deletion of RecQL5 in BLM Ϫ/Ϫ cells results in a further increase of the SCE frequency that is higher than that of BLM Ϫ/Ϫ cells (41). RecQL5 possesses a DNA helicase activity similar to that of BLM, which may explain their overlapping roles in SCE suppression. Both helicases have 3Ј-to-5Ј polarity and can promote branch migration for Holliday junctions (15), the displacement of D loops, and the disruption of Rad51 presynaptic filaments (20). However, R...

Background:The BRC repeat is essential for BRCA2 to bind RAD51 and promote homologous recombination. Results: A BRC repeat variant is essential for RECQL5 to bind RAD51 and suppress homologous recombination. Conclusion: The BRC repeat can be utilized to either promote or suppress homologous recombination. Significance: Discovery of multiple functions of the BRC repeat is important for understanding regulation of homologous recombination.