At first glance, the picture above might look like just a bunch of random doodles on your chemistry notes. But actually, these drawings represent the diversity of of morphologies and motility techniques found in the microbial world. Some bacteria are rod-shaped (bacillus), while others are curved (vibrioid), spiral (spirochete), or round (coccoid). While the mechanisms used by bacteria to form bacillus, vibrioid, and coccoid shapes are well understood, less is known about how spirochete bacteria achieve a helical structure. Some helical bacteria harbor flagella in between their inner and outer membranes that are essential for spiral shape. Other spiral bacteria, however, lack flagella but are able to maintain their shape using a yet unidentified mechanism.
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| A magnified view of Helicobacter pylori - scan microscopy, courtesy of Czech University of Life Science Prague |
One species of spirochete is Helicobacter pylori, which is a member of the Epsilonproteobacteria family. H. pylori is a helical bacterium that colonizes the stomach in about half of the world population when ingested orally. The bacteria can cause infection that may lead to gastric inflammation and cancer in a subset of those infected. It has been posited that the helical shape of H. pylori facilitates its motility within the thick mucus of the stomach, thus contributing to its pathogenicity. In recent years, Laura Sycuro and her team identified four genes that play a role in creating the cell elongation, curvature, and twist that produce H. pylori’s corkscrew-like shape. Cell shape is determined by its peptidoglycan (PG) layer. PG is composed of glycan (sugar) strands that are crosslinked by protein, forming a stable mesh structure (see figure below). Alteration of the PG cross links, regulated by the four proteins that Sycuro identified, create the unique helical shape of H. pylori.
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| Peptidoglycan cross-links are comprised of sugar strands (blue) which are positioned perpendicular to protein "bridges" (orange). Together they create a flexible mesh that is a component of the bacterial cell wall. |
The Sycuro Lab was able to identify the role of four cell-shape determining genes by cloning H. pylori and analyzing the mutant clones with shape defects. Following a visual screen of 2,000 H. pylori cells, they identified nine mutants, including one with a vibrioid (remember, this means curved rod) shape. This mutation is in the HPG27_1481 coding sequence, known as tagE, which has an active site homologous to the wild type bacteria. They then deleted the tagE sequence and observed the same curved-rod shape as the mutants. This data identified tagE as a crucial gene involved in determining helical rather than vibrioid morphology, and they named it csd1. They then identified a second gene upstream of csd1 on the same locus, meaning the genes are located at the same spot along the chromosome. When this second gene is deleted, it results in vibrioid cells similar to the csd1 mutants. They named this gene csd2. Targeted deletion of a third gene at this locus, ccmA, resulted in vibrioid cells as well. Together, these three genes, csd1, csd2, and ccmA, make up a locus that largely controls the morphology of H. pylori cells. The deletion of any single gene on this locus results in improper curvature and a loss of the helical twist observed in wild-type cells.
H. pylori strains encode a fourth cell-shape determining gene named csd3, found at a separate locus. Targeted deletion of this gene resulted in a variety of mutant morphologies, including highly-curved “c”-shaped cells, coccoid cells, and cells with little to no curvature. These tests suggest that csd3 is another gene involved in determining cell curvature and helical twist in H. pylori. The csd genes (1-3) and ccmA that the Sycuro Lab found to be integral in the formation of H. pylori helical shape are only found in bacteria with peptidase activity, even though the csd genes are found in other species. Peptidases regulate the degradation of proteins, suggesting that these genes may regulate the breakdown of proteins like those found in the PG crosslinks.
Images from Figure 1 of the Sycuro paper show the unique morphologies achieved by the mutant cells. Images B and C display wild-type cells while images D-G and I-L show the mutant phenotypes that Sycuro and her team observed. A clear loss of curvature and twist can be seen in cells with csd1, csd2, csd3, and ccmA knockout phenotypes.
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| Figure 1. Wild-type morphology (B, C) and mutant morphologies (D-G and I-L) in H. pylori. Image H shows the location of csd3 at a different locus from csd1, csd2, and ccmA. |
Sycuro and her team again looked at the isolated csd mutants and saw that the cell shape changes occur simultaneous with mutant peptidoglycan sacculi. The sacculus (plural sacculi) is a structural “sack” that retains the shape of the bacteria when the inside of the cell is removed. When the PG sacculi of H. pylori was isolated, they saw rods with varying curvature for csd1 and csd3 mutants, whereas they saw helices for the wild type. These data supports the idea that the structural determinant of cell morphology is the PG sacculus. The mutants exhibited changes in glycan strand connectivity associated with the cell curvature and twist, but did not show any weakening in the cell wall integrity or cell motility. This phenomenon is observed in bubbles that wobble and twist when they are first blown. The shape of the bubble changes and twists, but the structure of the bubble as whole is not compromised (until it pops of course). The data collected by the Sycuro lab supports the idea that relaxation of peptidoglycan facilitates cell curvature and, ultimately, helical formation.
The Sycuro Lab showed that csd mutations (1-3), as well as ccmA, are all required for the helical shape formation in H. pylori and that the PG sacculus is the structural determinant of cell shape. Based on these data, Sycuro and her team proposed a model for how H. pylori achieves the curvature and twist associated with the wild type helical morphology via peptidoglycan relaxation. The four cell-shape determining genes they identified act as peptidases, which as you may recall means they catalyze the hydrolysis (breakdown) of proteins. H. pylori must precisely localize these hydrolytic activities to the outer edge of the cell in order to achieve just the right shape. Sycuro proposes that the cell achieves curvature by hydrolyzing PG crosslinks and therefore relaxing the stiff glycan strands. To understand this mechanism, imagine your fingers are caught in a Chinese finger trap. In order to free your fingers, you must push in on the trap, relaxing the tension holding your fingers in place. Targeted hydrolysis of PG along a specific axis achieves the same goal. The relaxation of the glycan strands allows the cell wall more freedom of movement, resulting in curvature. In order to achieve twist, H. pylori shifts the axis diagonally and/or hydrolyzes PG at the point of inflection, which is marked by the pink stripe in the figure below.
Their next step was to look at the significance of cell shape on cell function; why do we care that H. pylori is this shape? H. pylori’s helical shape is associated with its pathogenicity, so they investigated the wild type and mutant cells’ ability to colonize the stomachs of mice. The helical wild type H. pylori outcompeted the mutant cells and fewer of the mutant cells colonized in the mice stomachs. The data Sycuro collected suggested that wild-type PG cross-linking promoted efficient stomach colonization, while those with more rigid PG cell walls had compromised colonization abilities.Sycuro concluded that the helical shape is important for pathogenesis, a phenomenon that has been observed in other pathogens. The helical shape allows H. pylori to infiltrate the stomach lining easily, similar to the physics of the cork screw turning into a wine bottle. If you’ve ever tried remove a cork from a wine bottle with a knife or other sharp object, I bet you wished you had a corkscrew. The helical shape of the corkscrew allows it to penetrate through the thick cork better than a straight object.
The Sycuro Lab showed that csd mutations (1-3), as well as ccmA, are all required for the helical shape formation in H. pylori and that the PG sacculus is the structural determinant of cell shape. Based on these data, Sycuro and her team proposed a model for how H. pylori achieves the curvature and twist associated with the wild type helical morphology via peptidoglycan relaxation. The four cell-shape determining genes they identified act as peptidases, which as you may recall means they catalyze the hydrolysis (breakdown) of proteins. H. pylori must precisely localize these hydrolytic activities to the outer edge of the cell in order to achieve just the right shape. Sycuro proposes that the cell achieves curvature by hydrolyzing PG crosslinks and therefore relaxing the stiff glycan strands. To understand this mechanism, imagine your fingers are caught in a Chinese finger trap. In order to free your fingers, you must push in on the trap, relaxing the tension holding your fingers in place. Targeted hydrolysis of PG along a specific axis achieves the same goal. The relaxation of the glycan strands allows the cell wall more freedom of movement, resulting in curvature. In order to achieve twist, H. pylori shifts the axis diagonally and/or hydrolyzes PG at the point of inflection, which is marked by the pink stripe in the figure below.
Image (top) is Figure 6. From Sycuro et al. Gif (below) generated from a video courtesy of Yakomoga
Their next step was to look at the significance of cell shape on cell function; why do we care that H. pylori is this shape? H. pylori’s helical shape is associated with its pathogenicity, so they investigated the wild type and mutant cells’ ability to colonize the stomachs of mice. The helical wild type H. pylori outcompeted the mutant cells and fewer of the mutant cells colonized in the mice stomachs. The data Sycuro collected suggested that wild-type PG cross-linking promoted efficient stomach colonization, while those with more rigid PG cell walls had compromised colonization abilities.Sycuro concluded that the helical shape is important for pathogenesis, a phenomenon that has been observed in other pathogens. The helical shape allows H. pylori to infiltrate the stomach lining easily, similar to the physics of the cork screw turning into a wine bottle. If you’ve ever tried remove a cork from a wine bottle with a knife or other sharp object, I bet you wished you had a corkscrew. The helical shape of the corkscrew allows it to penetrate through the thick cork better than a straight object.
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| Gif created from a video courtesy of Painting With a Twist. |
This research gets at the heart of bacterial cell shape and motility. It emphasizes a theme that is seen throughout biology: form dictates function. The function of specific genes within the H. pylori genome is important to the shape of the bacteria, which in turn dictates its pathogenicity. The work done by Sycuro and her collaborators is important in understanding how this pathogenic bacterium colonizes the stomach of humans, where it can cause ulcers and gastric cancer. The identification of four LytM peptidase homologs suggests the possibility of new treatment or prevention strategies that target these proteins. Understanding the role that H. pylori’s helical shape plays in its pathogenicity has implications for treatments that may prevent helix formation and thereby decrease its invasiveness and toxicity. This work has granted us a fuller understanding of the mechanisms cells use to achieve helical shape, which can be explored further in other spirochete species, especially those that may have pathogenic qualities.
Works Cited:
Sycuro, L.K.; Pincus, Z.; Gutierrez, K.D.; Biboy, J.; Stern, C. A.; Vollmer, W. and Salama, N. R. (2010). Peptidoglycan crosslinking relaxation promoted Helicobacter pylori's helical
shape and stomach colonization. Cell 141, 822-833.
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| Meet the authors: Megan Littlehale (left) and Isa Rodriguez (right) |
Megan Littlehale is a sophomore at Mount Holyoke College. She is a Biology and Philosophy double major from Reading, Massachusetts. Megan works in Professor Amy Camp’s research lab at MHC, which focuses on sporulation processes in Bacillus subtilis. She also volunteers at an adult ESL class in Chicopee, which is the highlight of her week. In her free time, she enjoys watching The Office and finding the best coffee in the Pioneer Valley.
Isa Rodriguez is a senior at Mount Holyoke College, majoring in Neuroscience and behavior. She is a member of the varsity lacrosse team, a director for Diversions a Cappella and serves as the reassure for the class of 2017. Following graduation she plans to go to dental school to be a pediatric dentist. In her free time she makes memes about Mount Holyoke and finds relatable GIFs for the everyday woman.
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