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Cell morphology as a virulence determinant: lessons from Helicobacter pylori
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ScienceDirect Cell morphology as a virulence determinant: lessons from Helicobacter pylori Nina R Salama A genetic screen for colonization factors of the human stomach pathogen Helicobacter pylori took a surprising turn with the discovery that some colonization mutants had lost helical cell morphology. Further pursuit of direct morphology screens revealed a large H. pylori ‘shapesome’ complex consisting of peptidoglycan modification and precursor synthesis enzymes, a cytoskeletal element and putative scaffold or regulatory proteins that promote enhanced asymmetric cell wall growth. Functional characterization of H. pylori shape mutants indicates multiple roles for cell shape during colonization of mucosal surfaces. Conservation of both the molecular constituents of the H. pylori cell shape program and a newly appreciated enrichment of this morphotype at mucosal surface suggests that helical organisms may be particularly well poised to exploit host perturbations to become pathogens. Address Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, Seattle, WA 98109, United States Corresponding author: Salama, Nina R ([email protected])
Current Opinion in Microbiology 2019, 48:11–17 This review comes from a themed issue on Stanley Falkow alumni Edited by Denise Monack and Igor Brodsky
https://doi.org/10.1016/j.mib.2019.12.002 1369-5274/ã 2020 Elsevier Ltd. All rights reserved.
Discovering shape as a virulence determinant
had heard that this recently discovered bacterium causes ulcers but was stunned when he showed me a preprint from the International Agency for Research on Cancer (IARC) that would declare H. pylori a carcinogen for its association with stomach cancer [1,2]. The opportunity to work on such an insidious pathogen, which must wield highly sophisticated weapons to cause these horrible chronic diseases, seemed too good to pass up. I soon discovered the perils of working with a new organism. Residing in the Epsilonproteobacteria class of the Proteobacteria phylum, H. pylori is as evolutionarily distant as you can get from well-studied enteric Proteobacteria like Escherichia coli. Thus, I spent most of my postdoc building tools (murine infection model, transposon library [3–5]) and only after moving to an independent position did we start genetic screens for pathogenesis [6]. While our screen identified genes in expected functional pathways (motility, urease), the largest category by far (81 of our 223 hits) had no functional annotations, suggesting that H. pylori might use different strategies for host colonization compared to other studied pathogens. An early investigator grant from the Pew Charitable Trusts provided the opportunity to meet with Christine Jacobs-Wagner, who discovered the intermediate filament protein crescentin as the key determinant of the curved-rod shape of Caulobacter crescentus [7]. Thanks to this meeting, one of my first graduate students decided to perform a limited microscopy-based screen of our transposon library for cell morphology mutants. Her initial screen of 2000 random clones gave three mutants: one straight, one curved, and one mostly straight with occasional bends. We mapped the straight mutant first and found that the transposon had hopped into HP1075, one of the hypothetical proteins in our colonization screen [6,8]. This emboldened us to start a new research program focusing on the role of helical cell morphology in H. pylori pathogenesis ultimately discovering that helical cell shape impacts both colonization and inflammation in the stomach.
When I wrote to Stanley Falkow back in 1994 as an almost-PhD yeast cell biologist, I told him that I wanted to leverage bacterial genetics as a probe to learn about human cell biology. This apparently resonated. According to Stan, “Bacteria are excellent cell biologists”. Little did I know that I would end up studying the cell biology of the bacterium (not the host) and find that bacterial cell morphology plays multiple roles in virulence.
H. pylori reveals new mechanisms of cell shape specification
While my inspiration for switching into the field of bacterial pathogenesis for postdoctoral work derived from exciting work on Salmonella and Yersinia Type III secretion system effectors, during my interview Falkow pitched a completely different bug: Helicobacter pylori. I
The three mutant clones from our initial screen led to the discovery of six cell shape-determining (csd) genes. The curved mutant, csd1, resides in a locus containing two neighboring genes, Csd2 and CcmA, that also promote helical cell shape [9]. Csd1, a predicted M23 family peptidase, has D,D-endopeptidase activity that cleaves
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Current Opinion in Microbiology 2020, 54:11–17
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the most abundant peptide crosslinks (tetra–pentapeptide crosslinks) which link the repeating disaccharide chains that give tensile strength to the peptidoglycan (PG) sacculus. Csd2, which is encoded upstream of csd1, also has homology to M23 peptidases, but lacks conservation of key active site residues. Subsequent work showed that Csd2 forms heterodimers with Csd1 that stabilize Csd1 protein levels [10,11]. H. pylori encodes a third M23 peptidase homologue that promotes helical cell shape, Csd3, which shows D,D-endopeptidase (breaking tetra–pentapeptide crosslinks) as well as weak D,D-carboxypeptidase (trimming monomeric pentapeptides to tetrapeptides) activity in vitro [9,12]. The gene downstream of csd1 encodes a cytosolic bactofilin called CcmA. Like other bactofilins [13,14,15], CcmA spontaneously forms polymers, polymer bundles and lattices in vitro that can be visualized by transmission electron microscopy [16,17]. The remaining straight and nearly straight mutant clones had disrupted Csd4, a new M14 subfamily D,L-carboxypeptidase that trims monomeric tripeptides to dipeptides [8,18], or Csd5, a putative single pass inner membrane scaffolding protein [8]. Additional genetic screens leveraged the altered light scattering properties of cell shape mutants that can be measured using flow cytometry to perform genome wide surveys to capture all the nonessential, nonredundant genes that promote helical cell shape [11,19]. These screens identified two more PG hydrolases (Csd6 and Slt) and another putative scaffolding protein (Csd7). Csd6 encodes an L,D-carboxypeptidase that cleaves monomeric tetrapeptides to tripeptides [19–21] and Slt encodes a exolytic lytic transglycosylase that cleaves between the repeating PG disaccharides [22]. Assembly of a parts list positioned us to explore how these proteins collectively promote helical cell shape. Cell shape in most bacteria, including H. pylori, derives from the shape of the PG sacculus, as seen in the observation that isolated sacculi retain the shape of the cell [9]. A helix has inherent asymmetry with a minor helical axis defined as the shortest helical path connecting the two poles and a longer major helical axis 180 degrees opposite the minor helical axis (Figure 1). The Csd proteins presumably work to impose a helical structure on the PG sacculus. While cocci can arise from uniform growth in all directions, many straight rods (bacilli) utilize a Rod complex. This complex contains a remote actin homologue, MreB, that biases the motion and localization of PG synthases along the side wall during cell elongation to enforce a straight-rod shape with minimal curvature and uniform diameter [23–26]. H. pylori has most components of the Rod complex, yet maintains substantial surface curvatures. Two other curved organisms, C. crescentus and Vibrio cholerae, only reveal their helicity after filamentation since their helical pitch (the distance for one complete helical turn) is longer than the cell length. These organisms impose Current Opinion in Microbiology 2020, 54:11–17
curvature through the action of cell-spanning intermediate filament-like cytoskeletal polymers (CreS and CrvA, respectively) that mechanically restrict synthesis locally, thus promoting synthesis on the opposite side of the cell [27,28]. While H. pylori has multiple predicted intermediate filament proteins (the coiled coil rich proteins or Ccrp), they do not appear to form cellspanning filaments and show strain variable modulation of cell shape [11,29]. The finding of multiple PG hydrolases in our cell shape screens raises the possibility that H. pylori uses a different mechanism(s) to promote the higher surface curvatures imposed by its helical cell shape. Possible mechanisms include localized remodeling of PG crosslinking to distort the straight cylinder and PG hydrolysis serving as a signal to stimulate asymmetric synthesis. As cell integrity under turgor pressure requires a continuity of the PG sacculus, PG hydrolases must be tightly regulated to prevent cell lysis. Furthermore, both hydrolase-centric models require spatially organized activity to create a regular helix. A combination of bacterial two hybrid, protein stability, and immunoprecipitation experiments in H. pylori cells, suggest the existence of one or more ‘shapesome’ complexes containing Csd proteins (Figure 1). The multipass inner membrane protein Csd7 links the periplasmic PG endopeptidase Csd1 and its stabilization partner Csd2 to CcmA polymers [11]. CcmA also copurifies with Csd5, which additionally binds PG and the cytoplasmic PG precursor synthesis enzyme MurF [30]. Localization of CcmA within cells using immunofluorescence revealed short puncta along the length of the cell enriched at surface Gaussian curvatures corresponding to the major helical axis [17]. Short pulses of cell wall metabolic probes indicate higher relative synthesis at these same Gaussian curvatures [17]. Interestingly, MreB showed relative depletion at the major helical axis suggesting this extra synthesis may be MreB independent. The cell wall synthesis enrichment results combined with the finding of a PG synthesis enzyme and hydrolases in CcmA-containing protein complexes appear to support localized hydrolase-dependent stimulation of synthesis at sites of positive Gaussian curvature (as opposed to restriction of synthesis on the opposite side of the cell) as one of the mechanisms H. pylori utilizes for helical cell shape maintenance. Remaining open questions include whether CcmA directly senses positive Gaussian curvature, or is recruited by a shapesome protein. Does the shapesome recognize a specific cell wall feature? How do the PG remodifying activities of cell shape proteins not detected in the shapesome complex purifications to date (Csd3, Csd4, Csd6, Slt) contribute to shape maintenance and how are they spatially regulated? Which synthases (RodA, PBP1) direct the enhanced PG synthesis at the major helical axis? www.sciencedirect.com
H. pylori cell morphology and virulence Salama 13
Figure 1
(a)
helical pitch
minor axis
major axis (b)
peptidoglycan
(c)
Slt Csd4 Csd5
Csd3 inner membrane
Csd6
Csd7
Csd2 Csd1
CcmA MurF Current Opinion in Microbiology
Helicobacter pylori cell shape mutants uncover a helical cell shape promoting ‘shapesome’ complex. (a) H. pylori, like all helical organisms, has a minor helical axis defined by the shortest helical path between the two poles and a longer major helical axis 180 degrees opposite. Helical pitch is the distance for one helical turn. (b) Transmission electron micrographs of representative mutants with three morphologies found in cell shape screens described in this review: variable (csd3, green), curved (csd1, red), straight (csd4, blue). Scale bar 1 mm. (c) Model of characterized enzymatic activities and protein-protein interactions among known H. pylori cell shape determining proteins (see text for details). Wedge cutouts indicate hydrolase activity and color coding indicates mutant morphologic phenotype as shown in (b).
Functional impact of helical shape: is motility in mucus the whole story? While we still have work to do in resolving the precise molecular mechanisms by which H. pylori builds a helix, our many shape mutants provide an opportunity to www.sciencedirect.com
rigorously explore the functional significance of cell shape for H. pylori stomach colonization and pathogenesis. With its small genome H. pylori encodes no redundancy in PG synthases; the single class A penicillin binding protein and core Rod complex members are essential [31,32,33]. Current Opinion in Microbiology 2020, 54:11–17
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Surprisingly, our cell shape mutants show normal growth rates, suggesting the cell growth and cell shape PG modification programs are distinct. Our cell shape mutants also have normal flagellation, allowing us to explore the major hypothesis in the field that helical cell shape enhances motility of H. pylori in mucus, thus facilitating access to the gastric epithelium. Throughout the GI tract, the mucus layer acts both as physical barrier to bacterial penetration and a scaffold for creating a gradient of antimicrobial compounds. H. pylori primarily localize to the stomach’s inner mucus layer and adheres to gastric epithelial cells. To probe how helical shape influences H. pylori’s movement in mucus, we used live cell tracking of isogenic helical wild-type and straight mutants in three different strain backgrounds to show that helical cell shape gives a modest (7–21%) increase in swimming speeds in physiologic concentrations of purified porcine gastric mucin [34]. A subsequent study, using higher magnification and image acquisition frame rates to capture flagellar bundle position and rotation frequencies as well cell body rotation frequencies and precession angles, calculated a 15% enhancement of propulsive thrust by a helical cell body compared to a straight mutant [35]. While effects of helical cell shape on swimming speeds appear subtle, loss of helical shape also increased the fraction of bacteria completely immobilized in mucin by 30–40%, which could influence the rate at which turnover of the mucus layer clears bacteria [34]. Assessment of penetration into and growth within gastric glands during infection of a mouse stomach colonization model indicated modest attenuation for a straight mutant (csd6) at one week post-infection [36]. After one month the population of wild-type (helical) bacteria localized within gastric glands had contracted, while the straight mutant expanded within the glands. Paradoxically, inflammatory scores at one and three months post-infection were lower for the straight mutant in comparison to wild-type, in spite of higher bacterial burden within the glands. These results contrast with similar work in Campylobacter jejuni where the failure of two different straight mutants to induce inflammation appears secondary to penetration into the intestinal crypts [37]. H. pylori straight mutants may interact with gastric epithelial cells in the glands less efficiently than wild-type helical cells, although in co-culture with gastric cell lines in vitro, straight mutants induce similar levels of proinflammatory cytokines as wild-type [8,9]. A recent study could suggest an alternative explanation. Suarez et al. showed that activation of the Nod1 pathway, one of the major innate immune pathways activated by H. pylori, results in a net immunosuppressive phenotype, perhaps through altered macrophage polarization [38]. Considering this immunosuppressive activity, the higher gland burden of the straight mutants may drive development of an immunosuppressive Current Opinion in Microbiology 2020, 54:11–17
environment in the glands through Nod1 activation that allows further expansion of the gland population. Work utilizing tissue clearing and co-infection of strains marked with different fluorescent protein markers suggests a lack of mixing between the gland and surface mucus populations [39]. However, H. pylori residing in glands can clonally expand to neighboring glands. Helical shape could be particularly important for promoting survival in the inner mucous layer if cells immobilized in mucus more rapidly get cleared by mucus sloughing and peristalsis. One limitation of the H. pylori mouse stomach colonization studies is the failure to visualize the inner mucous layer population of bacteria (due to difficulty preserving the mucus during tissue sectioning), and thus the inability to assess the proportion of bacteria residing within the glands versus in the inner mucous layer. Since mouse infection cannot yet be monitored in real time, further elucidation of interactions among gland and surface mucus subpopulations will require development of better tissue models, which could include organoids grown on three dimensional scaffolds and/or tissue slice cultures [40].
Conservation of Csds and helical shape as a pathogenesis strategy Throughout his career, Falkow pondered characteristics of pathogens that distinguish them from commensal bacteria [41–43]. Pathogens, he said, have the feature of intimately interacting with and/or breaching the epithelium. Helical cell shape promotes colonization of the gastric glands and inner mucus providing the opportunity to interact with epithelial cells. Thus, helical cell shape does appear to fit the criteria of a virulence factor. To stay in this host tissue-proximal environment, pathogens must also tolerate oxygen and antimicrobial innate and adaptive immune defenses. For H. pylori, it appears that helical cell shape may also influence some of these other pathogenic properties. In the years since our first study showing stomach colonization defects for H. pylori cell shape mutants [9,12], shape-associated colonization defects have been observed for C. jejuni [37,44,45] and V. cholerae [27]. Homologues of the Csd PG modifying enzymes, which exist throughout the Epsilonproteobacteria and Deltaproteobacteria, have been extensively characterized in C. jejuni, where they show similar shape phenotypes. A small molecule inhibitor targeting the active site of Csd4 from H. pylori and Pgp 1 from C. jejuni perturbs cell shape in both organisms [46]. Additionally, a bactofilin homologue alters helical cell pitch in the Spirochete Leptospira biflexa [47]. Mining of microbiome surveys in monkeys, mice, and humans that compared the luminal and mucosa-associated bacterial communities revealed an enrichment of organisms with spiral/helical cell shape amongst mucosa-associated organisms [48,49,50]. Could mucosa-associated helical organisms be poised www.sciencedirect.com
H. pylori cell morphology and virulence Salama 15
to turn pathogenic? One of the few Deferribacteres phylum members of mammalian microbiomes, Mucospirillum schaedleri, has helical cell shape, and its genome sequence reveals homologues to all four H. pylori characterized Csd PG modifying enzymes as well as a CcmA/ bactofilin homologue [51]. M. schaedleri localizes to the colonic epithelial surface and has been shown to confer protection from Salmonella enterica serovar Typhimurium tissue invasion and colitis, but not Salmonella luminal colonization [48]. In the context of combined mutation of two Crohn’s disease-associated mutations (NOD2 and phagocyte NADPH oxidase subunit CYBB), M. schaedleri induces spontaneous colitis [52]. Thus, this normally commensal helical organism can become an opportunistic pathogen. The role of cell morphology in M. schaedleri niche occupation and pathogenic potential seems worth exploring. While helical shape correlates with intimate association with mucosal surfaces, it could confer some fitness costs. H. pylori clinical isolates show a range of helical parameters with some showing nearly straight morphology. Moreover, in C. jejuni straight variants arise at high frequency through phase variation of pgp1 and pgp2 [53–56]. For C. jejuni, passage through chick embryos selects for straight morphology [57] and a recent study found enrichment of morphology variants and an excess of rare mutations in mreB, pgp1 and pgp2 in extraintestinal isolates compared to gastroenteritis isolates [58]. The finding in the mouse model that straight shape might be advantageous for H. pylori in the gland environment could indicate selection for different cell morphologies during different phases of infection. Investigation of correlations between clinical outcome and cell morphology in H. pylori may begin to shed light on this possibility.
Conflict of interest statement Nothing declared.
Acknowledgements I thank members of the Salama lab for helpful comments and Jennifer A. Taylor for figure preparation. Research reported here was supported in part by US National Institutes of HealthR01 AI136946 and U01 CA221230.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions and outlook Bacterial genetics led me on an incredibly rewarding journey studying bacterial cell biology rather than host cell biology. This journey required me to develop a more nuanced view of bacterial pathogenesis and overcome a rather naı¨ve intuition that bacteria have ‘simple’ morphology. Bacteria require incredibly diverse and complex mechanisms for survival in pathogenic niches. We now understand that bacterial morphology should be included in this arsenal. A key to my group’s success in this area derives from an excellent piece of Falkow advice: look in the microscope. I’m always surprised at how few microbiologists actually look at their cultures. To quote Yogi Berra, as Falkow often did, “You can observe a lot by just watching.” Luckily there is still much to learn. I look forward to watching how the work in my lab and excellent work from others will elucidate both the precise biophysical mechanisms that H. pylori and other helical organisms use to shape a helical cell wall and the specific functional aspects of host-pathogen interactions influenced by bacterial cell morphology. www.sciencedirect.com
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34. Martinez LE, Hardcastle JM, Wang J, Pincus Z, Tsang J, Hoover TR, Bansil R, Salama NR: Helicobacter pylori strains vary cell shape and flagellum number to maintain robust motility in viscous environments. Mol Microbiol 2016, 99:88-110.
20. Kim HS, Im HN, An DR, Yoon JY, Jang JY, Mobashery S, Hesek D, Lee M, Yoo J, Cui M et al.: The cell shape-determining Csd6 protein from Helicobacter pylori constitutes a new family of L, D-carboxypeptidase. J Biol Chem 2015, 290:25103-25117.
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36. Martinez LE, O’Brien VP, Leverich CK, Knoblaugh SE, Salama NR: Nonhelical Helicobacter pylori mutants show altered gland colonization and elicit less gastric pathology than helical bacteria during chronic infection. Infect Immun 2019, 87 Detailed imaging combined with histology during an extended time course of murine stomach infection suggests that straight muants show altered immune induction in spite of robust gland colonizaiton. These results suggest pathogenic functions for helical cell shape beyond motility in mucus.
22. Chaput C, Labigne A, Boneca IG: Characterization of Helicobacter pylori lytic transglycosylases Slt and MltD. J Bacteriol 2007, 189:422-429. 23. Bratton BP, Shaevitz JW, Gitai Z, Morgenstein RM: MreB polymers and curvature localization are enhanced by RodZ and predict E. coli’s cylindrical uniformity. Nat Commun 2018, 9:2797. 24. Hussain S, Wivagg CN, Szwedziak P, Wong F, Schaefer K, Izore T, Renner LD, Holmes MJ, Sun Y, Bisson-Filho AW et al.: MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis. eLife 2018, 7:e32471. 25. Ursell TS, Nguyen J, Monds RD, Colavin A, Billings G, Ouzounov N, Gitai Z, Shaevitz JW, Huang KC: Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proc Natl Acad Sci U S A 2014, 111: E1025-1034. 26. Wong F, Garner EC, Amir A: Mechanics and dynamics of translocating MreB filaments on curved membranes. eLife 2019, 8:e40472. 27. Bartlett TM, Bratton BP, Duvshani A, Miguel A, Sheng Y, Martin NR, Nguyen JP, Persat A, Desmarais SM, VanNieuwenhze MS et al.: A periplasmic polymer curves Vibrio cholerae and promotes pathogenesis. Cell 2017, 168:172185 e115 This study identified CrvA, which is the first known periplasmic intermediant filament like protein. CrvA promotes higher relative rates of cell wall synthesis on the opposite side of the cell leading to cell curvature revealing a cell shape mechanism similar yet distinct to that in C. crecentus. Mutant crvA strains show reduced colonization and pathogenesis in two different infection models linking cell shape with pathogenesis of V. cholerae for the first time. Current Opinion in Microbiology 2020, 54:11–17
37. Stahl M, Frirdich E, Vermeulen J, Badayeva Y, Li X, Vallance BA, Gaynor EC: The helical shape of Campylobacter jejuni promotes in vivo pathogenesis by aiding transit through intestinal mucus and colonization of crypts. Infect Immun 2016, 84:3399-3407. 38. Suarez G, Romero-Gallo J, Piazuelo MB, Sierra JC, Delgado AG, Washington MK, Shah SC, Wilson KT, Peek RM Jr: Nod1 imprints inflammatory and carcinogenic responses toward the gastric pathogen Helicobacter pylori. Cancer Res 2019, 79:1600-1611 Nod1 mutation in two different H. pylori murine disease models indicates a net immunospressive function for H. pylori activation of the Nod1 pathway, which may result from altered macrophage polarization. Thus while Nod1 is generally thought to activate inflammation and promote clearance of infection, H. pylori may subvert this pathway by a yet to be described mechanism. 39. Fung C, Tan S, Nakajima M, Skoog EC, Camarillo-Guerrero LF, Klein JA, Lawley TD, Solnick JV, Fukami T, Amieva MR: Highresolution mapping reveals that microniches in the gastric glands control Helicobacter pylori colonization of the stomach. PLoS Biol 2019, 17:e3000231. 40. Dutton JS, Hinman SS, Kim R, Wang Y, Allbritton NL: Primary cellderived intestinal models: recapitulating physiology. Trends Biotechnol 2019, 37:744-760. 41. Falkow S: What is a pathogen? Am Soc Microbiol News 1997, 63:359-365. 42. Falkow S: The microbe’s view of infection. Ann Intern Med 1998, 129:247-248. www.sciencedirect.com
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43. Finlay BB, Falkow S: Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 1997, 61: 136-169. 44. Frirdich E, Biboy J, Adams C, Lee J, Ellermeier J, Gielda LD, Dirita VJ, Girardin SE, Vollmer W, Gaynor EC: Peptidoglycanmodifying enzyme Pgp1 is required for helical cell shape and pathogenicity traits in Campylobacter jejuni. PLoS Pathog 2012, 8:e1002602. 45. Frirdich E, Vermeulen J, Biboy J, Soares F, Taveirne ME, Johnson JG, DiRita VJ, Girardin SE, Vollmer W, Gaynor EC: Peptidoglycan LD-carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the DLcarboxypeptidase Pgp1. J Biol Chem 2014, 289:8007-8018. 46. Liu Y, Frirdich E, Taylor JA, Chan AC, Blair KM, Vermeulen J, Ha R, Murphy ME, Salama NR, Gaynor EC et al.: A bacterial cell shapedetermining inhibitor. ACS Chem Biol 2016, 11:981-991. 47. Jackson KM, Schwartz C, Wachter J, Rosa PA, Stewart PE: A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa. Mol Microbiol 2018, 108:77-89. 48. Herp S, Brugiroux S, Garzetti D, Ring D, Jochum LM, Beutler M, Eberl C, Hussain S, Walter S, Gerlach RG et al.: Mucispirillum schaedleri antagonizes salmonella virulence to protect mice against colitis. Cell Host Microbe 2019, 25:681-694 e688 Detailed analysis of both mucosal and luminal associated bacterial populations reveals that this minor component of the microbiome confers resistance to systemic Salmonella infection and associated colitis, but not luminal colonization. Mining existing human data suggests common (42%) colonization of the human interstinal mucosa even though M. schaedleri is rarely detected in feces. Thus our understanding of the importance of helical organisms in human health may be underappreciated from fecal sampling of the microbiome. 49. Yasuda K, Oh K, Ren B, Tickle TL, Franzosa EA, Wachtman LM, Miller AD, Westmoreland SV, Mansfield KG, Vallender EJ et al.: Biogeography of the intestinal mucosal and lumenal microbiome in the rhesus macaque. Cell Host Microbe 2015, 17:385-391. 50. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, Kotler E, Zur M, Regev-Lehavi D, Brik RB et al.: Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell 2018, 174:1388-1405.e21.
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51. Loy A, Pfann C, Steinberger M, Hanson B, Herp S, Brugiroux S, Gomes Neto JC, Boekschoten MV, Schwab C, Urich T et al.: Lifestyle and horizontal gene transfer-mediated evolution of Mucispirillum schaedleri, a core member of the murine gut microbiota. mSystems 2017, 2. 52. Caruso R, Mathes T, Martens EC, Kamada N, Nusrat A, Inohara N, Nunez G: A specific gene-microbe interaction drives the development of Crohn’s disease-like colitis in mice. Sci Immunol 2019, 4 Using mice doubly mutant for two known Chrohn’s disease associated mutations this study shows that M. shaedleri can cause spontaneous colitis. Generalizing this result could suggest that helical organisms, by virtue of intimate association with the epithelial surface, are poised to become pathogenic during host perturbation. 53. Esson D, Gupta S, Bailey D, Wigley P, Wedley A, Mather AE, Meric G, Mastroeni P, Sheppard SK, Thomson NR et al.: Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape. Microb Pathog 2017, 104:202-211. 54. Esson D, Mather AE, Scanlan E, Gupta S, de Vries SP, Bailey D, Harris SR, McKinley TJ, Meric G, Berry SK et al.: Genomic variations leading to alterations in cell morphology of Campylobacter spp. Sci Rep 2016, 6:38303. 55. Frirdich E, Biboy J, Huynh S, Parker CT, Vollmer W, Gaynor EC: Morphology heterogeneity within a Campylobacter jejuni helical population: the use of calcofluor white to generate rodshaped C. jejuni 81-176 clones and the genetic determinants responsible for differences in morphology within 11168 strains. Mol Microbiol 2017, 104:948-971. 56. Gaynor EC, Cawthraw S, Manning G, MacKichan JK, Falkow S, Newell DG: The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulenceassociated phenotypes. J Bacteriol 2004, 186:503-517. 57. Field LH, Underwood JL, Payne SM, Berry LJ: Characteristics of an avirulent Campylobacter jejuni strain and its virulenceenhanced variants. J Med Microbiol 1993, 38:293-300. 58. Wheeler NE, Blackmore T, Reynolds AD, Midwinter AC, Marshall J, French NP, Savoian MS, Gardner PP, Biggs PJ: Genomic correlates of extraintestinal infection are linked with changes in cell morphology in Campylobacter jejuni. Microb Genom 2019, 5.
Current Opinion in Microbiology 2020, 54:11–17
ScienceDirect Cell morphology as a virulence determinant: lessons from Helicobacter pylori Nina R Salama A genetic screen for colonization factors of the human stomach pathogen Helicobacter pylori took a surprising turn with the discovery that some colonization mutants had lost helical cell morphology. Further pursuit of direct morphology screens revealed a large H. pylori ‘shapesome’ complex consisting of peptidoglycan modification and precursor synthesis enzymes, a cytoskeletal element and putative scaffold or regulatory proteins that promote enhanced asymmetric cell wall growth. Functional characterization of H. pylori shape mutants indicates multiple roles for cell shape during colonization of mucosal surfaces. Conservation of both the molecular constituents of the H. pylori cell shape program and a newly appreciated enrichment of this morphotype at mucosal surface suggests that helical organisms may be particularly well poised to exploit host perturbations to become pathogens. Address Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, Seattle, WA 98109, United States Corresponding author: Salama, Nina R ([email protected])
Current Opinion in Microbiology 2019, 48:11–17 This review comes from a themed issue on Stanley Falkow alumni Edited by Denise Monack and Igor Brodsky
https://doi.org/10.1016/j.mib.2019.12.002 1369-5274/ã 2020 Elsevier Ltd. All rights reserved.
Discovering shape as a virulence determinant
had heard that this recently discovered bacterium causes ulcers but was stunned when he showed me a preprint from the International Agency for Research on Cancer (IARC) that would declare H. pylori a carcinogen for its association with stomach cancer [1,2]. The opportunity to work on such an insidious pathogen, which must wield highly sophisticated weapons to cause these horrible chronic diseases, seemed too good to pass up. I soon discovered the perils of working with a new organism. Residing in the Epsilonproteobacteria class of the Proteobacteria phylum, H. pylori is as evolutionarily distant as you can get from well-studied enteric Proteobacteria like Escherichia coli. Thus, I spent most of my postdoc building tools (murine infection model, transposon library [3–5]) and only after moving to an independent position did we start genetic screens for pathogenesis [6]. While our screen identified genes in expected functional pathways (motility, urease), the largest category by far (81 of our 223 hits) had no functional annotations, suggesting that H. pylori might use different strategies for host colonization compared to other studied pathogens. An early investigator grant from the Pew Charitable Trusts provided the opportunity to meet with Christine Jacobs-Wagner, who discovered the intermediate filament protein crescentin as the key determinant of the curved-rod shape of Caulobacter crescentus [7]. Thanks to this meeting, one of my first graduate students decided to perform a limited microscopy-based screen of our transposon library for cell morphology mutants. Her initial screen of 2000 random clones gave three mutants: one straight, one curved, and one mostly straight with occasional bends. We mapped the straight mutant first and found that the transposon had hopped into HP1075, one of the hypothetical proteins in our colonization screen [6,8]. This emboldened us to start a new research program focusing on the role of helical cell morphology in H. pylori pathogenesis ultimately discovering that helical cell shape impacts both colonization and inflammation in the stomach.
When I wrote to Stanley Falkow back in 1994 as an almost-PhD yeast cell biologist, I told him that I wanted to leverage bacterial genetics as a probe to learn about human cell biology. This apparently resonated. According to Stan, “Bacteria are excellent cell biologists”. Little did I know that I would end up studying the cell biology of the bacterium (not the host) and find that bacterial cell morphology plays multiple roles in virulence.
H. pylori reveals new mechanisms of cell shape specification
While my inspiration for switching into the field of bacterial pathogenesis for postdoctoral work derived from exciting work on Salmonella and Yersinia Type III secretion system effectors, during my interview Falkow pitched a completely different bug: Helicobacter pylori. I
The three mutant clones from our initial screen led to the discovery of six cell shape-determining (csd) genes. The curved mutant, csd1, resides in a locus containing two neighboring genes, Csd2 and CcmA, that also promote helical cell shape [9]. Csd1, a predicted M23 family peptidase, has D,D-endopeptidase activity that cleaves
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Current Opinion in Microbiology 2020, 54:11–17
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the most abundant peptide crosslinks (tetra–pentapeptide crosslinks) which link the repeating disaccharide chains that give tensile strength to the peptidoglycan (PG) sacculus. Csd2, which is encoded upstream of csd1, also has homology to M23 peptidases, but lacks conservation of key active site residues. Subsequent work showed that Csd2 forms heterodimers with Csd1 that stabilize Csd1 protein levels [10,11]. H. pylori encodes a third M23 peptidase homologue that promotes helical cell shape, Csd3, which shows D,D-endopeptidase (breaking tetra–pentapeptide crosslinks) as well as weak D,D-carboxypeptidase (trimming monomeric pentapeptides to tetrapeptides) activity in vitro [9,12]. The gene downstream of csd1 encodes a cytosolic bactofilin called CcmA. Like other bactofilins [13,14,15], CcmA spontaneously forms polymers, polymer bundles and lattices in vitro that can be visualized by transmission electron microscopy [16,17]. The remaining straight and nearly straight mutant clones had disrupted Csd4, a new M14 subfamily D,L-carboxypeptidase that trims monomeric tripeptides to dipeptides [8,18], or Csd5, a putative single pass inner membrane scaffolding protein [8]. Additional genetic screens leveraged the altered light scattering properties of cell shape mutants that can be measured using flow cytometry to perform genome wide surveys to capture all the nonessential, nonredundant genes that promote helical cell shape [11,19]. These screens identified two more PG hydrolases (Csd6 and Slt) and another putative scaffolding protein (Csd7). Csd6 encodes an L,D-carboxypeptidase that cleaves monomeric tetrapeptides to tripeptides [19–21] and Slt encodes a exolytic lytic transglycosylase that cleaves between the repeating PG disaccharides [22]. Assembly of a parts list positioned us to explore how these proteins collectively promote helical cell shape. Cell shape in most bacteria, including H. pylori, derives from the shape of the PG sacculus, as seen in the observation that isolated sacculi retain the shape of the cell [9]. A helix has inherent asymmetry with a minor helical axis defined as the shortest helical path connecting the two poles and a longer major helical axis 180 degrees opposite the minor helical axis (Figure 1). The Csd proteins presumably work to impose a helical structure on the PG sacculus. While cocci can arise from uniform growth in all directions, many straight rods (bacilli) utilize a Rod complex. This complex contains a remote actin homologue, MreB, that biases the motion and localization of PG synthases along the side wall during cell elongation to enforce a straight-rod shape with minimal curvature and uniform diameter [23–26]. H. pylori has most components of the Rod complex, yet maintains substantial surface curvatures. Two other curved organisms, C. crescentus and Vibrio cholerae, only reveal their helicity after filamentation since their helical pitch (the distance for one complete helical turn) is longer than the cell length. These organisms impose Current Opinion in Microbiology 2020, 54:11–17
curvature through the action of cell-spanning intermediate filament-like cytoskeletal polymers (CreS and CrvA, respectively) that mechanically restrict synthesis locally, thus promoting synthesis on the opposite side of the cell [27,28]. While H. pylori has multiple predicted intermediate filament proteins (the coiled coil rich proteins or Ccrp), they do not appear to form cellspanning filaments and show strain variable modulation of cell shape [11,29]. The finding of multiple PG hydrolases in our cell shape screens raises the possibility that H. pylori uses a different mechanism(s) to promote the higher surface curvatures imposed by its helical cell shape. Possible mechanisms include localized remodeling of PG crosslinking to distort the straight cylinder and PG hydrolysis serving as a signal to stimulate asymmetric synthesis. As cell integrity under turgor pressure requires a continuity of the PG sacculus, PG hydrolases must be tightly regulated to prevent cell lysis. Furthermore, both hydrolase-centric models require spatially organized activity to create a regular helix. A combination of bacterial two hybrid, protein stability, and immunoprecipitation experiments in H. pylori cells, suggest the existence of one or more ‘shapesome’ complexes containing Csd proteins (Figure 1). The multipass inner membrane protein Csd7 links the periplasmic PG endopeptidase Csd1 and its stabilization partner Csd2 to CcmA polymers [11]. CcmA also copurifies with Csd5, which additionally binds PG and the cytoplasmic PG precursor synthesis enzyme MurF [30]. Localization of CcmA within cells using immunofluorescence revealed short puncta along the length of the cell enriched at surface Gaussian curvatures corresponding to the major helical axis [17]. Short pulses of cell wall metabolic probes indicate higher relative synthesis at these same Gaussian curvatures [17]. Interestingly, MreB showed relative depletion at the major helical axis suggesting this extra synthesis may be MreB independent. The cell wall synthesis enrichment results combined with the finding of a PG synthesis enzyme and hydrolases in CcmA-containing protein complexes appear to support localized hydrolase-dependent stimulation of synthesis at sites of positive Gaussian curvature (as opposed to restriction of synthesis on the opposite side of the cell) as one of the mechanisms H. pylori utilizes for helical cell shape maintenance. Remaining open questions include whether CcmA directly senses positive Gaussian curvature, or is recruited by a shapesome protein. Does the shapesome recognize a specific cell wall feature? How do the PG remodifying activities of cell shape proteins not detected in the shapesome complex purifications to date (Csd3, Csd4, Csd6, Slt) contribute to shape maintenance and how are they spatially regulated? Which synthases (RodA, PBP1) direct the enhanced PG synthesis at the major helical axis? www.sciencedirect.com
H. pylori cell morphology and virulence Salama 13
Figure 1
(a)
helical pitch
minor axis
major axis (b)
peptidoglycan
(c)
Slt Csd4 Csd5
Csd3 inner membrane
Csd6
Csd7
Csd2 Csd1
CcmA MurF Current Opinion in Microbiology
Helicobacter pylori cell shape mutants uncover a helical cell shape promoting ‘shapesome’ complex. (a) H. pylori, like all helical organisms, has a minor helical axis defined by the shortest helical path between the two poles and a longer major helical axis 180 degrees opposite. Helical pitch is the distance for one helical turn. (b) Transmission electron micrographs of representative mutants with three morphologies found in cell shape screens described in this review: variable (csd3, green), curved (csd1, red), straight (csd4, blue). Scale bar 1 mm. (c) Model of characterized enzymatic activities and protein-protein interactions among known H. pylori cell shape determining proteins (see text for details). Wedge cutouts indicate hydrolase activity and color coding indicates mutant morphologic phenotype as shown in (b).
Functional impact of helical shape: is motility in mucus the whole story? While we still have work to do in resolving the precise molecular mechanisms by which H. pylori builds a helix, our many shape mutants provide an opportunity to www.sciencedirect.com
rigorously explore the functional significance of cell shape for H. pylori stomach colonization and pathogenesis. With its small genome H. pylori encodes no redundancy in PG synthases; the single class A penicillin binding protein and core Rod complex members are essential [31,32,33]. Current Opinion in Microbiology 2020, 54:11–17
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Surprisingly, our cell shape mutants show normal growth rates, suggesting the cell growth and cell shape PG modification programs are distinct. Our cell shape mutants also have normal flagellation, allowing us to explore the major hypothesis in the field that helical cell shape enhances motility of H. pylori in mucus, thus facilitating access to the gastric epithelium. Throughout the GI tract, the mucus layer acts both as physical barrier to bacterial penetration and a scaffold for creating a gradient of antimicrobial compounds. H. pylori primarily localize to the stomach’s inner mucus layer and adheres to gastric epithelial cells. To probe how helical shape influences H. pylori’s movement in mucus, we used live cell tracking of isogenic helical wild-type and straight mutants in three different strain backgrounds to show that helical cell shape gives a modest (7–21%) increase in swimming speeds in physiologic concentrations of purified porcine gastric mucin [34]. A subsequent study, using higher magnification and image acquisition frame rates to capture flagellar bundle position and rotation frequencies as well cell body rotation frequencies and precession angles, calculated a 15% enhancement of propulsive thrust by a helical cell body compared to a straight mutant [35]. While effects of helical cell shape on swimming speeds appear subtle, loss of helical shape also increased the fraction of bacteria completely immobilized in mucin by 30–40%, which could influence the rate at which turnover of the mucus layer clears bacteria [34]. Assessment of penetration into and growth within gastric glands during infection of a mouse stomach colonization model indicated modest attenuation for a straight mutant (csd6) at one week post-infection [36]. After one month the population of wild-type (helical) bacteria localized within gastric glands had contracted, while the straight mutant expanded within the glands. Paradoxically, inflammatory scores at one and three months post-infection were lower for the straight mutant in comparison to wild-type, in spite of higher bacterial burden within the glands. These results contrast with similar work in Campylobacter jejuni where the failure of two different straight mutants to induce inflammation appears secondary to penetration into the intestinal crypts [37]. H. pylori straight mutants may interact with gastric epithelial cells in the glands less efficiently than wild-type helical cells, although in co-culture with gastric cell lines in vitro, straight mutants induce similar levels of proinflammatory cytokines as wild-type [8,9]. A recent study could suggest an alternative explanation. Suarez et al. showed that activation of the Nod1 pathway, one of the major innate immune pathways activated by H. pylori, results in a net immunosuppressive phenotype, perhaps through altered macrophage polarization [38]. Considering this immunosuppressive activity, the higher gland burden of the straight mutants may drive development of an immunosuppressive Current Opinion in Microbiology 2020, 54:11–17
environment in the glands through Nod1 activation that allows further expansion of the gland population. Work utilizing tissue clearing and co-infection of strains marked with different fluorescent protein markers suggests a lack of mixing between the gland and surface mucus populations [39]. However, H. pylori residing in glands can clonally expand to neighboring glands. Helical shape could be particularly important for promoting survival in the inner mucous layer if cells immobilized in mucus more rapidly get cleared by mucus sloughing and peristalsis. One limitation of the H. pylori mouse stomach colonization studies is the failure to visualize the inner mucous layer population of bacteria (due to difficulty preserving the mucus during tissue sectioning), and thus the inability to assess the proportion of bacteria residing within the glands versus in the inner mucous layer. Since mouse infection cannot yet be monitored in real time, further elucidation of interactions among gland and surface mucus subpopulations will require development of better tissue models, which could include organoids grown on three dimensional scaffolds and/or tissue slice cultures [40].
Conservation of Csds and helical shape as a pathogenesis strategy Throughout his career, Falkow pondered characteristics of pathogens that distinguish them from commensal bacteria [41–43]. Pathogens, he said, have the feature of intimately interacting with and/or breaching the epithelium. Helical cell shape promotes colonization of the gastric glands and inner mucus providing the opportunity to interact with epithelial cells. Thus, helical cell shape does appear to fit the criteria of a virulence factor. To stay in this host tissue-proximal environment, pathogens must also tolerate oxygen and antimicrobial innate and adaptive immune defenses. For H. pylori, it appears that helical cell shape may also influence some of these other pathogenic properties. In the years since our first study showing stomach colonization defects for H. pylori cell shape mutants [9,12], shape-associated colonization defects have been observed for C. jejuni [37,44,45] and V. cholerae [27]. Homologues of the Csd PG modifying enzymes, which exist throughout the Epsilonproteobacteria and Deltaproteobacteria, have been extensively characterized in C. jejuni, where they show similar shape phenotypes. A small molecule inhibitor targeting the active site of Csd4 from H. pylori and Pgp 1 from C. jejuni perturbs cell shape in both organisms [46]. Additionally, a bactofilin homologue alters helical cell pitch in the Spirochete Leptospira biflexa [47]. Mining of microbiome surveys in monkeys, mice, and humans that compared the luminal and mucosa-associated bacterial communities revealed an enrichment of organisms with spiral/helical cell shape amongst mucosa-associated organisms [48,49,50]. Could mucosa-associated helical organisms be poised www.sciencedirect.com
H. pylori cell morphology and virulence Salama 15
to turn pathogenic? One of the few Deferribacteres phylum members of mammalian microbiomes, Mucospirillum schaedleri, has helical cell shape, and its genome sequence reveals homologues to all four H. pylori characterized Csd PG modifying enzymes as well as a CcmA/ bactofilin homologue [51]. M. schaedleri localizes to the colonic epithelial surface and has been shown to confer protection from Salmonella enterica serovar Typhimurium tissue invasion and colitis, but not Salmonella luminal colonization [48]. In the context of combined mutation of two Crohn’s disease-associated mutations (NOD2 and phagocyte NADPH oxidase subunit CYBB), M. schaedleri induces spontaneous colitis [52]. Thus, this normally commensal helical organism can become an opportunistic pathogen. The role of cell morphology in M. schaedleri niche occupation and pathogenic potential seems worth exploring. While helical shape correlates with intimate association with mucosal surfaces, it could confer some fitness costs. H. pylori clinical isolates show a range of helical parameters with some showing nearly straight morphology. Moreover, in C. jejuni straight variants arise at high frequency through phase variation of pgp1 and pgp2 [53–56]. For C. jejuni, passage through chick embryos selects for straight morphology [57] and a recent study found enrichment of morphology variants and an excess of rare mutations in mreB, pgp1 and pgp2 in extraintestinal isolates compared to gastroenteritis isolates [58]. The finding in the mouse model that straight shape might be advantageous for H. pylori in the gland environment could indicate selection for different cell morphologies during different phases of infection. Investigation of correlations between clinical outcome and cell morphology in H. pylori may begin to shed light on this possibility.
Conflict of interest statement Nothing declared.
Acknowledgements I thank members of the Salama lab for helpful comments and Jennifer A. Taylor for figure preparation. Research reported here was supported in part by US National Institutes of HealthR01 AI136946 and U01 CA221230.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 61: Schistosomes, liver flukes, and Helicobacter pylori. Lyon: IARC: World Health Organization International Association for Research on Cancer; 1994.
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Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD: Helicobacter pylori infection and gastric lymphoma. N Engl J Med 1994, 330:1267-1271.
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Salama N, Guillemin K, McDaniel TK, Sherlock G, Tompkins L, Falkow S: A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc Natl Acad Sci U S A 2000, 97:14668-14673.
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Salama NR, Otto G, Tompkins L, Falkow S: Vacuolating cytotoxin of Helicobacter pylori plays a role during colonization in a mouse model of infection. Infect Immun 2001, 69:730-736.
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Salama NR, Shepherd B, Falkow S: Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J Bacteriol 2004, 186:7926-7935.
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Baldwin DN, Shepherd B, Kraemer P, Hall MK, Sycuro LK, PintoSantini DM, Salama NR: Identification of Helicobacter pylori genes that contribute to stomach colonization. Infect Immun 2007, 75:1005-1016.
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Sycuro LK, Wyckoff TJ, Biboy J, Born P, Pincus Z, Vollmer W, Salama NR: Multiple peptidoglycan modification networks modulate Helicobacter pylori’s cell shape, motility, and colonization potential. PLoS Pathog 2012, 8:e1002603.
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Sycuro LK, Pincus Z, Gutierrez KD, Biboy J, Stern CA, Vollmer W, Salama NR: Peptidoglycan crosslinking relaxation promotes Helicobacter pylori’s helical shape and stomach colonization. Cell 2010, 141:822-833.
Conclusions and outlook Bacterial genetics led me on an incredibly rewarding journey studying bacterial cell biology rather than host cell biology. This journey required me to develop a more nuanced view of bacterial pathogenesis and overcome a rather naı¨ve intuition that bacteria have ‘simple’ morphology. Bacteria require incredibly diverse and complex mechanisms for survival in pathogenic niches. We now understand that bacterial morphology should be included in this arsenal. A key to my group’s success in this area derives from an excellent piece of Falkow advice: look in the microscope. I’m always surprised at how few microbiologists actually look at their cultures. To quote Yogi Berra, as Falkow often did, “You can observe a lot by just watching.” Luckily there is still much to learn. I look forward to watching how the work in my lab and excellent work from others will elucidate both the precise biophysical mechanisms that H. pylori and other helical organisms use to shape a helical cell wall and the specific functional aspects of host-pathogen interactions influenced by bacterial cell morphology. www.sciencedirect.com
10. An DR, Im HN, Jang JY, Kim HS, Kim J, Yoon HJ, Hesek D, Lee M, Mobashery S, Kim SJ et al.: Structural basis of the heterodimer formation between cell shape-determining proteins Csd1 and Csd2 from Helicobacter pylori. PLoS One 2016, 11:e0164243. 11. Yang DC, Blair KM, Taylor JA, Petersen TW, Sessler T, Tull CM, Leverich CK, Collar AL, Wyckoff TJ, Biboy J et al.: A genome-wide Helicobacter pylori morphology screen uncovers a membrane-spanning helical cell shape complex. J Bacteriol 2019, 201 Using flow cytometry to measure perturbations in light scattering the study defined the full set of non-essential nonredundant genes required for H. pylori helical cell morphology. 12. Bonis M, Ecobichon C, Guadagnini S, Prevost MC, Boneca IG: A M23B family metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence. Mol Microbiol 2010, 78:809-819. 13. Deng X, Gonzalez Llamazares A, Wagstaff JM, Hale VL, Cannone G, McLaughlin SH, Kureisaite-Ciziene D, Lowe J: The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity. Nat Microbiol 2019, 12:2357-2368 Current Opinion in Microbiology 2020, 54:11–17
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Using a combination of X-ray crystallography and cryo-electron microscopy this study showed that bactofillins form head-to-head and tail-totail polymers and can induce curvature of membranes.
28. Cabeen MT, Charbon G, Vollmer W, Born P, Ausmees N, Weibel DB, Jacobs-Wagner C: Bacterial cell curvature through mechanical control of cell growth. EMBO J 2009, 28:1208-1219.
14. Kuhn J, Briegel A, Morschel E, Kahnt J, Leser K, Wick S, Jensen GJ, Thanbichler M: Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus. EMBO J 2011, 29: 327-339.
29. Waidner B, Specht M, Dempwolff F, Haeberer K, Schaetzle S, Speth V, Kist M, Graumann PL: A novel system of cytoskeletal elements in the human pathogen Helicobacter pylori. PLoS Pathog 2009, 5:e1000669.
15. Vasa S, Lin L, Shi C, Habenstein B, Riedel D, Kuhn J, Thanbichler M, Lange A: beta-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR. Proc Natl Acad Sci U S A 2015, 112:E127-136. 16. Holtrup S, Heimerl T, Linne U, Altegoer F, Noll F, Waidner B: Biochemical characterization of the Helicobacter pylori bactofilin-homolog HP1542. PLoS One 2019, 14:e0218474. 17. Taylor JA, Bratton BP, Sichel SR, Blair KM, Jacobs HM, DeMeester KE, Kuru E, Gray J, Biboy J, VanNieuwenhze MS et al.: Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori. Elife 2020, 9:e52482 Using 3D reconstructions and super resolution microscopy this study indicates a role for the CcmA bactofilin in promoting enhanced cell wall synthesis at the major helical axis to sustain helical cell shape. 18. Chan AC, Blair KM, Liu Y, Frirdich E, Gaynor EC, Tanner ME, Salama NR, Murphy ME: Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4. J Biol Chem 2015, 290:3622-3638.
30. Blair KM, Mears KS, Taylor JA, Fero J, Jones LA, Gafken PR, Whitney JC, Salama NR: The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton. Mol Microbiol 2018, 110:114-127. 31. Boneca IG, Ecobichon C, Chaput C, Mathieu A, Guadagnini S, Prevost MC, Colland F, Labigne A, de Reuse H: Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori. Appl Environ Microbiol 2008, 74:2095-2102. 32. Contreras-Martel C, Martins A, Ecobichon C, Trindade DM, Mattei PJ, Hicham S, Hardouin P, Ghachi ME, Boneca IG, Dessen A: Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex. Nat Commun 2017, 8:776 Co-crystal structures of PBP2 and MreC reveal that H. pylori utilizes the Rod complex for cell elongation, similar to straight-rod shaped organisms. 33. El Ghachi M, Mattei PJ, Ecobichon C, Martins A, Hoos S, Schmitt C, Colland F, Ebel C, Prevost MC, Gabel F et al.: Characterization of the elongasome core PBP2: MreC complex of Helicobacter pylori. Mol Microbiol 2011, 82:68-86.
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