Princeton researchers find the key to piercing the armor of harmful bacteria

Bacteria are single-celled organisms essential to human health, both in our environment and within our body. However, some bacterial species can make us sick.

When a doctor suspects a disease of bacterial origin, they will perform diagnostic tests to identify which bacterial species is causing the disease so that a course of treatment can be devised. One such test is called a Gram stain, named after Hans Christian Gram, who developed the technique in the 1880s. Gram found that some bacterial species, so-called “Gram-negative” bacteria, shrug off a purple dye. he was using to help visualize microbes under a microscope. Scientists eventually found that Gram-negative bacteria resist absorption of the dye because they are enveloped in what is, essentially, a microbial armor: their vulnerable cell membrane is protected by a tightly packed layer of sugars called the cell wall. , and moreover, a specialized outer membrane.

“Understanding how bacteria build this barrier is an important step in engineering strategies to bypass it,” said Thomas Silhavy, professor of molecular biology at Warner-Lambert Parke-Davis and senior author of two new papers studying the outer membrane, one in the Proceedings of the National Academy of Sciences and the other in Trends in Microbiology.

One of the main components of the outer membrane is a unique molecule called lipopolysaccharide (LPS), which covers the surface of the cell. “LPS helps increase the mechanical strength of the Gram-negative cell envelope and also forms a surface coating that prevents toxic molecules, including some antibiotics, from entering the cell,” said Randi Guest, a postdoctoral researcher. associate in the Silhavy laboratory, professor of molecular biology and lead author of the article Trends.

LPS is a notoriously potent toxin that can cause serious illness when released from the bacterial outer membrane or secreted from the cell.

“The amount of LPS produced by the cell is carefully controlled, as too little LPS can lead to cell rupture, while too much LPS, especially if not properly assembled, is toxic,” said Guest. “We looked at studies of three essential membrane proteins that monitor not only LPS biosynthesis within the cell, but also transport and proper assembly on the cell surface.”

As discussed by Guest and colleagues in their paper, the construction of the bacterial outer membrane poses a complex problem for bacteria because potentially dangerous LPS, produced inside the cell, must be transported through the cell wall to reach the outer membrane. Furthermore, these processes must be balanced with the production and transport of the other components of the membrane, which in Gram-negative bacteria is mainly made up of a class of molecules called phospholipids.

“A long-standing mystery in the field is how phospholipids are transported to the outer membrane,” Silhavy said. One idea is that phospholipids can passively flow back and forth between the bacterium’s inner cell membrane and its outer membrane in the contact zones, but this idea is highly controversial. New research from Silhavy’s group lends support to the idea that there is a passive mode of transport.

Jackie Grimm, then a graduate student in Silhavy’s lab, along with Handuo Shi, a graduate student in KC Huang’s lab at Stanford, led an effort to identify proteins involved in the trafficking of phospholipids between inner and outer membranes. For their studies, colleagues used bacteria that have a mutation that increases the rate at which phospholipids flow from the inner to the outer membrane. When deprived of nutrients, these bacteria undergo shrinking and rupture of the inner membrane, followed by cell death, because they are unable to produce new phospholipids for the inner membrane to replace those lost in the outer membrane. The authors introduced further mutations into these bacteria, then looked for genes that, when mutated, affect how quickly the bacteria die after nutrient withdrawal.

“We used next-generation sequencing to screen for genes involved in this process and found that gene disruption a slowed the transport of phospholipids, “Silhavy said.

Although their data indicate that the protein encoded by a involved in the transport of phospholipids between the inner cell membrane and the outer membrane, Grimm, Shi and their colleagues noted that it is not yet clear how the YhdP protein works to influence this process. A potential clue could be found in its predicted similarity to other proteins whose function is already known. One of these is a mammalian protein that forms a channel that carries phospholipids across membranes.

“This suggests that YhdP might form a hydrophobic channel between the inner and outer membrane through which phospholipids flow,” Silhavy noted.

“Silhavy and colleagues provide the strongest data to date to identify how phospholipids are transported between membranes in bacteria, an elusive question for decades in our field,” said M. Stephen Trent, Distinguished Professor of Infectious Diseases at the University of Georgia who was not involved in the work. “They make a strong argument with genetics and biophysics that a protein of unknown function, YhdP, influences a rapid process of phospholipid transport between membranes. It will be really interesting to learn about the role of YhdP in phospholipid transport in the future.”

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Quotes:

Randi L. Guest, Steven T. Rutherford and Thomas J. Silhavy. Border control: regulation of LPS biogenesis. Trends in microbiology. (2020) doi: https: //doi.org /10.1016 /j.tim.2020.09.008

Jacqueline Grimm, Handuo Shi, Wei Wang, Angela M. Mitchell, Ned S. Wingreen, Kerwyn Casey Huang, and Thomas J. Silhavy. The inner membrane protein YhdP modulates the flow rate of antegrade phospholipids Escherichia coli. Proceedings of the National Academy of Sciences, UNITED STATES OF AMERICA. (2020) doi: https: //doi.org /10.1073 /pnas.2015556117

Funding: Work in the Silhavy laboratory is supported by the NIGMS grant 5R35GM118024 (to TJS). To research a He was also supported by the NIGMS T32-GM007388 Scholarship (to JG), and in part by NIH Grant R01 GM082938 (to NSW), a James McDonnell Postdoctoral Scholarship (to HS). KCH is a Chan Zuckerberg Biohub Investigator.