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Advancing towards improved drug therapies for tuberculosis.

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Dibucaine specifically abrogates subpolar enrichment of IMD proteins in M. smegmatis. (a) Cells expressing mCherry-GlfT2 and Ppm1-mNeonGreen from internal sites were incubated with or without dibucaine for 9 h, OD600 The CFU was determined every 3 hours in biological triplicates. Means and standard deviations or individual data points are shown. (b) Effect of dibucaine on IMD-related proteins. The IMD marker proteins, mCherry-GlfT2 and Ppm1-mNeonGreen, were observed hourly during dibucaine treatment (left). Representative images from biological triplicates are shown. Scale bar = 5 µm. The size of the fluorescence intensity profiles along the long axis of the cells was measured using Oufti (right). Pg 1: n = 237 (before), 121 (1 hour), 289 (2 hours), 172 (3 hours). GlfT2: n= 127 (before), 119 (1 hour), 287 (2 hours), or 169 (3 hours). (C) The polar localization of DivIVA, a PM-CW marker protein, was examined before and after dibucaine treatment. Representative images of biological replicates are shown. n = 36 (before) or 37 (3 hours). (d) Sucrose gradient fractionation of cell lysates from the strain expressing mCherry-GlfT2 and Ppm1-mNeonGreen. The strain was treated with or without dibucaine. Ppm1-mNeonGreen (indicated by asterisks) was visualized by in-gel fluorescence after SDS-PAGE. Fluorescence intensity for each band was quantified and displayed in a bar graph. The antibody to PimB′ occasionally detects a cytoplasmic protein that migrates slightly less frequently than PimB, and we do not know the nature of this protein (8). PimB′ and MptC (indicated by asterisks) were visualized by western blotting using anti-PimB and rabbit anti-MptC antibodies. PimB′, IMD marker; MptC, a PM-CW marker, that was not affected by dibucaine treatment. Representative results from biological replicates are shown. (e) Recovery from dibucaine treatment. The same strain expressing mCherry-GlfT2 and Ppm1-mNeonGreen was treated with dibucaine for 3 h, washed and recovered in fresh Middlebrook 7H9 medium. Growth recovery was monitored by OD600. Time 0 corresponds to the beginning of the recovery period. Representative results from biological replicates are shown. credit: Mbeo(2023). DOI: 10.1128/mbio.03396-22

In ongoing research aimed at developing more effective treatments for tuberculosis (TB), microbiologists at the University of Massachusetts Amherst have identified a long-requested gene that plays a critical role in the growth and survival of the TB pathogen.

The discovery provides a potential target for drug therapies for a deadly disease that has few effective treatments, and in 2021 alone sickened 10.6 million people worldwide and caused 1.6 million deaths, according to the World Health Organization.

Published in the journal MbeoThe research showed that the putative cfa gene encodes an essential enzyme that is directly involved in the first step of the formation of tuberculostearic acid (TBSA), a fatty acid unique to the cell membranes of mycobacteria. TBSA was first isolated from mycobacteria nearly 100 years ago but exactly how it was synthesized has remained elusive.

“There is a long history associated with this very remarkable fatty acid,” says senior author Yasuo Morita, associate professor of microbiology, whose lab was conducted by lead authors Malavika Prithviraj and Takehiro Kado.

Experiments have revealed how TBSA controls the functions of the fungal plasma membrane, which acts as a protective barrier for tuberculosis pathogens to survive in the human host for decades.

“Cfa is directly involved in the formation of tuberculostearic acid and is also involved in the regulation of the plasma membrane, all of which fell into place with our hypothesis,” says Prithviraj.

The focus of the research in Morita’s lab is identifying ways to interrupt homeostasis in the thick, waxy cell envelope, which includes the plasma membrane, so mycobacteria are unable to grow or vulnerable to attack. Prithviraj, Ph.D. The student and his colleagues performed a cellular lipid analysis to confirm what researchers suspected some 60 years ago.

“People have been very, very interested in understanding how this lipid is made and what it does in the cell,” Morita says. “Malavika discovered that Cfa is the enzyme that makes this lipid, and it is such a unique lipid that researchers are pursuing this lipid as a diagnostic marker for tuberculosis.”

In previous experiments, Morita’s lab had noted that plasma membrane domains located in the polar regions of the cell are important for the growth of mycobacteria.

“We were interested in understanding how this particular membrane domain is divided and regulated in bacteria,” says Prithviraj. “We worked with a deletion strain of cfa and also a complementary strain where we could add it back into the bacteria and investigate its exact function.”

The TB pathogen usually remains alive but dormant in the body for years or decades, thanks to its protective surface structure. Morita and his team are working on a primarily non-pathogenic model organism to learn what traits the bacteria need for survival and growth.

The researchers found that TBSA also prevents “tight packing” within the membrane. “If the membrane is too stiff, it won’t function properly, so membrane dynamics, or maintaining membrane fluidity, is very important,” Morita says. “What we’ve shown in this paper is that tuberculostearic acid is likely to be a very important molecular switch for maintaining this proper fluidity.”

The findings will help researchers take the next step towards developing new treatments for tuberculosis.

“We would be interested in understanding the effects of the gene in TB infection and how Cfa might help the bacteria survive in the human host,” says Prithviraj. “If we find a way to disrupt membrane fluidity maintenance, cells will not be able to grow efficiently and will eventually die.”

“There are many drugs used to treat tuberculosis, but there was no previous evidence that this particular aspect of mycobacterial physiology could be used as a direct target,” Morita adds. “This study shows that it could be.”

more information:
Malavika Prithviraj et al, Tuberculostearic acid controls fungal membrane division, Mbeo(2023). DOI: 10.1128/mbio.03396-22

Journal information:
Mbeo


Provided by the University of Massachusetts Amherst


the quote: One Step Closer to Better Drug Therapies for Tuberculosis (2023, April 18) Retrieved April 18, 2023 from https://phys.org/news/2023-04-closer-drug-therapies-tuberculosis.html

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