Kiran, U. | bioRxiv Science

bioRxiv2023

Dissecting the Ca2+ dependence of Mycobacterium tuberculosis DesA1 function

Mycobacterium tuberculosis (M. tb) has a complex cell wall, largely composed of mycolic acids and long-chain fatty acids that play a crucial role in maintaining its integrity and permeability. This complex lipid structure has a role in abrogating the process of phagosome-lysosome fusion and infection establishment. The M. tb desaturase A1 (DesA1) catalyzes the introduction of position-specific double bonds, a key step in the biosynthesis of a diverse range of mycolic acids. We have previously demonstrated that M. tb DesA1 is a Ca2+-binding protein, belonging to the extended {beta}{gamma}-crystallin superfamily. Using a combination of biophysical and genetic approaches, we investigated the structural and functional significance of Ca2+ binding on DesA1 activity. A protein unfolding assay of the protein in the presence and absence of Ca2+ shows that Ca2+ binding imparts structural stability to DesA1. To identify the role of Ca2+, we introduced mutations at key residues in the identified Ca2+-binding motif of DesA1 and generated F303A, E304Q, and F303A-E304Q variants of DesA1. We identified F303 as a hot point which disables the protein for Ca2+ binding. Two other mutations E304Q and F303A-E304Q showed reduced Ca2+ binding. Complementation of a conditionally complemented desA1 deletion mutant strain of Mycobacterium smegmatis with these mutants, either failed to complement its growth phenotype or led to a compromise in complementation. In addition, the F303A and F303A-E304Q complements exhibit increased sensitivity to isoniazid, a first-line anti-tubercular drug, pointing to a cell wall permeability defect in these strains. Our findings highlight the critical importance of Ca2+ in the functioning of DesA1 and its implicit role in the maintenance of mycobacterial cellular integrity.

microbiology↗

bioRxiv2022

Status of susceptibility of the visceral leishmaniasis vector, Phlebotomus argentipes (Diptera: Psychodidae: Phlebotominae), to insecticides used for vector control in Nepal

BackgroundVisceral leishmaniasis (VL) is targeted for elimination as a public health problem in Nepal by 2023. For nearly three decades, the core vector control intervention in Nepal has been indoor residual spraying (IRS) with pyrethroids. Considering the long-term use of pyrethroids and possible development of resistance of the vector Phlebotomus argentipes sand flies, we monitored susceptibility status of their field populations to the insecticides of different classes, in villages with and without IRS activities in recent years. Methodology/Principal findingsSand flies were collected from villages with and without IRS in five VL endemic districts from August 2019 to November 2020. The WHO susceptibility test procedure was adopted using filter papers impregnated at the discriminating concentrations of insecticides of the following classes: pyrethroids (alpha-cypermethrin 0.05%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%), carbamates (bendiocarb 0.1%) and organophosphates (malathion 5%). Pyrethroid resistance intensity bioassays with papers impregnated with 5x of the discriminating concentrations, piperonyl butoxide (PBO) synergist-pyrethroid bioassays and DDT cross resistance bioassays were also performed. In the IRS villages, the vector sand flies were resistant (mortality rate <90%) to alpha-cypermethrin and possibly resistant (mortality rate 90-97%) to deltamethrin and lambda-cyhalothrin, while susceptibility to these insecticides was variable in the non-IRS villages. The vector was fully susceptible to bendiocarb and malathion in all villages. A delayed knockdown time (KDT50) with pyrethroids was observed in all villages. The pyrethroid resistance intensity was low, and the susceptibility improved at 5x of the discriminating concentrations. Enhanced pyrethroid susceptibility after pre-exposure to PBO and the DDT-pyrethroid cross-resistance were evident. Conclusions/SignificanceOur investigation showed that P. argentipes sand flies have emerged with pyrethroid resistance, suggesting the need to switch to alternative classes of insecticides such as organophosphates for IRS. We strongly recommend for the regular and systematic monitoring of insecticide resistance in sand flies to optimize the efficiency of vector control interventions to sustain VL elimination efforts in Nepal. Author summaryVisceral leishmaniasis (VL), transmitted by P. argentipes sand flies, is endemic in South-East Asian countries such as Bangladesh, India and Nepal, and is on the verge of elimination as a public health problem in Nepal by 2023. As part of the WHO Global Vector Control Response, entomological surveillance including insecticide resistance monitoring is one of the four main pillars of this strategy. In the early 1990s, the historical use of DDT for sand fly vector control was replaced with deltamethrin or alpha-cypermethrin, which have now been in use for almost three decades in Nepal. Suspecting that this long-term use of pyrethroids might have selected resistance in sand fly populations which would jeopardize control efforts, we conducted the first comprehensive survey to generate contemporary evidence of insecticide resistance in Nepal. For this, we performed WHO susceptibility tests in five VL endemic districts and found strong evidence of pyrethroid resistance in vector populations from the areas receiving IRS. Resistance mechanisms involved would probably be kdr mutations and monooxygenase. This study also endorses regular insecticide resistance monitoring to inform evidence-based decisions on insecticide use for vector control and to maintain the effectiveness of vector control measures as a core intervention in the fight against VL.

zoology↗

bioRxiv2020

Improved and Simplified Diagnosis of Covid-19 using TE Extraction from Dry Swabs

Rigorous testing is the way forward to fight the Covid-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer and economical by bypassing the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to current most widely used method. We also provide a variant of the new method that increases the efficiency of detection by about 20% compared to the currently used method. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.

molecular biology↗