The shapeshifting Helicobacter pylori: From a corkscrew to a ball

There is growing evidence that bacterial morphology is closely related to their lifestyle. The helical Helicobacter pylori relies on its unique shape for survival and efficient colonization of the human stomach. Yet, they have been observed to transform into another distinctive morphology, the spherical coccoid. Despite being hypothesized to be involved in the persistence and transmission of this species, years of effort in deciphering the roles of the coccoid form remain fruitless since contrasting observations regarding its lifestyle were reported. Here, we discuss the two forms of H. pylori with a focus on the coccoid form, the molecular mechanism behind its morphological transformation, and experimental approaches to further develop our understanding of this phenomenon. We also propose a putative mechanism of the coccoid formation in H. pylori through induction of a type‐I toxin‐antitoxin (TA) system recently shown to influence the morphology of this species.


| INTRODUC TI ON
Helicobacter pylori is a microaerophilic ε-proteobacterium that is the major cause of gastritis, peptic ulcers, and gastric cancer (Bray et al., 2018).In 2015, over half of the world's population was infected, and it was subsequently designated a high priority for antibiotic research and development by the World Health Organization in 2017 given its increase in antibiotic resistance (Davies, n.d.;Hooi et al., 2017).Their flagellar-driven motility and capacity to adhere to host cells as well as their virulence factors, CagA and VacA, are together essential for survival in the human stomach.Additionally, the helical shape of H. pylori has been shown to facilitate their movement inside the stomach by allowing them to bore into the mucus layer via a corkscrew-like motion, thereby escaping from the acidic environment and enhancing their colonization of the niche (Bonis et al., 2010;Martínez et al., 2019).Besides the helical shape, H. pylori can adopt and transition into spherical coccoid forms.While the significance of the helical form has been illustrated, the importance of the latter remains poorly understood (Bode et al., 1993;Enroth et al., 1999;Kusters et al., 1997;Sörberg et al., 1996;Willén et al., 2000).
In many bacterial species, morphological transformation is a manifestation of their survival strategy in unfavorable conditions.
Vibrio cholerae was among the first of several species of clinical interest to have been demonstrated to be able to transform from rod to coccoid shape when entering a reversible state of low metabolic activity called dormancy in which cells can persist for extended periods of time without dividing (Baker et al., 1983;Kondo et al., 1994;Kurath & Morita, 1983;Rollins & Colwell, 1986).Authors of these studies were also among the first to pinpoint the limitations of using plating method as a tool for assessing viability of bacterial cells as they observed that some bacteria continued to survive in marine environments despite having a drastic decline in colony number on plates.Accordingly, the term viable but non-culturable (VBNC) was adopted to describe bacterial cells in this condition; a state in which bacteria exhibit a detectable metabolic activity but cannot be cultured using standard laboratory techniques and is often preceded by a morphological transformation (Colwell et al., 1985;Kaprelyants & Kell, 1992;Oliver, 2005;Roszak et al., 1984;Xu et al., 1982).
The most worrisome concern related to the VBNC state of bacteria is its association with persistence, antibiotic tolerance, and immune evasion, as some bacterial species have been shown to undergo morphological transition when exposed to stressful conditions reminiscent of VBNC formation (Bode et al., 1993;Kusters et al., 1997;van Teeseling et al., 2017).For instance, H. pylori was found to transform from the helical to coccoid form when exposed to sub-MIC level of β-lactams such as amoxicillin (DeLoney & Schiller, 1999).
Coccoid H. pylori has been shown to evade recognition by the human Nod1 innate immune receptors and fails to induce neither NF-κB in HEK293T cells nor IL-8 production by gastric epithelial cells (Chaput et al., 2006).In a review encompassing 30 years of research on the viable but non-culturable state of bacteria, Pinto et al. pointed out the severe lack of studies on the mechanism of VBNC formation despite decades of research on the topic and how this could hinder us from tackling antibiotic resistance issues (Pinto et al., 2015).In a more recent study, stringent response, general stress response and toxin-antitoxin (TA) systems were presented as the most probable pathways for this phenomenon.Yet, the authors agreed that there is insufficient understanding on these topics to directly link formation mechanism of VBNC to bacterial persistence hence more detailed mechanistic studies are needed (Cai et al., 2022).
Bacterial cell shape is largely dictated by peptidoglycan (PGN) or cell wall; a heteropolymer consisted of glycan chains cross-linked together by peptide stems (Vollmer et al., 2008).Thus, it is very likely that the PGN biosynthesis and remodeling pathways are heavily involved in the morphological transformation of bacteria entering the VBNC state.Investigations targeting these processes could unravel the secrets behind this fascinating phenomenon.Nonetheless, this is a challenging task since bacteria have many redundant enzymes involved in the PGN pathways.For example, Escherichia coli has six D,D-endopeptidases (enzymes which hydrolyze D,D-crosslink of the peptide stems), three of which are redundantly essential for cell elongation in this bacterium and depletion of these enzymes leads to cell lysis (Egan et al., 2020;Singh et al., 2012).For this reason, H. pylori has been proposed as a model for studying morphological transformation in bacteria since this species has a minimal set of genes involved in PGN assembly and metabolism (Boneca et al., 2003; see Figure 1 for our current knowledge on PGN metabolism in H. pylori).
Additionally, this organism has been shown to transition from the helical to coccoid form in a similar manner that has been observed in other bacteria when entering the VBNC state.Thus, understanding the coccoid form of H. pylori as well as the molecular mechanism behind its formation could be our key to comprehending the molecular mechanism of VBNC formation in all bacteria including its significance to the public health.
This review provides an overview on the coccoid form of H. pylori with a critical analysis on important studies pertaining to its viability.Additionally, this review will explore the molecular mechanism of this morphological transformation by consolidating existing knowledge on the topic to provide an up-to-date overview while highlighting key players believed to be central in this process.Lastly, we propose a unique mechanism of coccoid formation in H. pylori through induction of a type-I toxin-antitoxin (TA) system that was recently shown to influence the morphology of this species.

| CO CCOID FORM OF H . pylo ri: DORMAN C Y OR DEG ENER ATION?
Upon its discovery by Barry Marshall in 1982 as a cause of gastritis and ulcers, the human stomach was initially thought to be the unique niche and reservoir of Helicobacter pylori.This notion followed unsuccessful attempts by Marshall and his team in developing an animal model for the specie, which he then took the liberty to experiment on himself and showed development of gastritis after ingesting a pure culture of the bacteria (Kyle et al., 2016;Marshall et al., 1985;Marshall & Warren, 1984).However, H. pylori had not been isolated from the environment by any standard laboratory culture techniques.It was not until 1989 that this species was shown to be able to survive in water for several months with decreased culturability while undergoing a morphological transition from spirals to curved rods, then to horse-shoe shapes and finally to spherical coccoids (Shahamat et al., 1989).This observation sparked speculation about its role in transmission as at that time, a few other gramnegative pathogens had been shown to also undergo morphological transformation into a round shape when entering dormancy (Kurath & Morita, 1983;Roszak et al., 1984;Xu et al., 1982).
A study on the importance of viable but non-culturable state reported VBNC in at least 85 bacterial species and among them, 51 were pathogenic to humans including H. pylori (Li et al., 2014).This is intriguing because whether the coccoid form of this species is the morphological manifestation of its degenerative state or is the VBNC form remains a controversy within the scientific community.Two of the many factors contributing to this debate are the dispute on their viability and the lack of clear evidence pertaining to its resuscitation.Some argued that the coccoid form of H. pylori is the manifestation of cell death due to an observed decrease in their genetic and cellular contents, lack of stage-specific proteins, and loss of membrane potential (Bumann et al., 2004;Enroth et al., 1999;Willén et al., 2000).Others have challenged this and proposed that the coccoid cells to be its persistent or dormant form but there exists no gold standard method to definitively confirm its viability (Bode et al., 1993;Pinto et al., 2015;Sörberg et al., 1996;Willén et al., 2000).Yet, there is also a third side to this argument as several studies observed at least two subtypes within the coccoid population of H. pylori; many other studies also came across a similar phenomenon but failed to grasp the significance of the distinction (Bode et al., 1993;Cellini et al., 2001;Kusters et al., 1997;Sato et al., 2003).
Several studies within the past few decades reported having successfully resuscitated H. pylori while many others failed (Aktas et al., 2020;Andersen et al., 1997;Cellini et al., 1994Cellini et al., , 1998;;Kurokawa et al., 1999;Richards et al., 2011).This task has proved to be extremely difficult both in vitro and in vivo, and many within the scientific community remain skeptical of the communicated results.
For instance, when 20-day-old H. pylori coccoids were used to infect mice, they were found to revert to the helical form and colonize the stomach with observed histopathological changes in the gastric mucosa (Cellini et al., 1994).However, some argue that morphological transformation in H. pylori is an asymptotic process and therefore a culture of pure coccoid form can never be obtained.Accordingly, the observations could have resulted from the few helical forms that remained among coccoid majority (Kusters et al., 1997).

| Dormancy
To explore the possible role of coccoid H. pylori in the transmission of this species, the viability of this life form was assessed at various levels.Structurally, these coccoid cells were found to be intact when examined using transmission electron microscopy (TEM).Their cell F I G U R E 1 Enzymatic actions of cell shape determinants in Helicobacter pylori.Once the lipid II precursor reaches the periplasm, it gets incorporated into the growing chain of peptidoglycan through mediation of glycosyltransferases such as PBP1 creating a glycosidic linkage between the NAG moiety of the lipid II and the NAM moiety of the nascent peptidoglycan.PBP1, PBP2 and PBP3 transpeptidases can all catalyze formation of a peptide bond between the m-DAP (position 3) of one peptide stem and the D-Ala (position 4) of another peptide stem.This peptide bond linking two peptide stems can in turn be cleaved by both the Csd1:Csd2 complex or the D,D-endopeptidase domain of Csd3/ HdpA.The D,D-carboxypeptidase domain of Csd3/HdpA cleaves the bond in between D-Ala (position 5) and D-Ala (position 4) within a peptide stem.Csd6 D,L-carboxypeptidase cleaves in between D-Ala (position 4) and m-DAP (position 3) and Csd4 L,D-carboxypeptidase cleaves in between m-DAP (position 3) and D-Glu (position 2).The AmiA amidase removes the peptide stem at L-Ala (position 1) from the NAM moiety of the glycan chain.Slt and MltD can both hydrolyze the glycosidic bond connecting NAM and NAG of the glycan chain.membrane, cytoplasm, and flagella were intact even after 3 months of storage.Distinct populations of the coccoid forms differentiable by size were reported when comparing effects of factors inducing coccoid formation in H. pylori.Those induced by amoxicillin produced a "mini form of coccoids" with a mean diameter of 0.3 μm compared with the average mean diameter of 0.8-1.0μm of those formed in stationary phase.Additionally, coccoid cells with distinct morphologies had also been observed within the same conditions.For example, examination of 5-day-old H. pylori coccoids from clinical strains using both Scanning Electron Microscopy (SEM) and TEM revealed two types of coccoid forms: coccoid cells with intact membranous structures and degenerated coccoids with fragmented membranes (Willén et al., 2000).A noteworthy observation in these cells was the presence of electron-dense aggregates inside their cytoplasm.They were identified to be polyphosphate granules and hypothesized to be a reservoir of energy and phosphorus supply during dormancy.
Further investigation into these electron-dense granules revealed that their accumulation was preceded by separation of the inner and outer membranes and followed by gradual migration toward the center of cells (Bode et al., 1993;Kusters et al., 1997;Willén et al., 2000).
DNA synthesis is the hallmark of an active life cycle in bacteria as new genetic material is needed for replication.However, this is not the case for bacterial cells in dormant state as they are nonreplicative and maintain only the basal level of cellular activities for survival.Nonetheless, active DNA synthesis has been reported in H. pylori coccoids.When 3-month-old coccoid cells were transferred into a liquid culture medium containing the thymidine analog Bromodeoxyuridine (BrdU), they were able to utilize it to synthesize new DNA materials as evidenced by the presence of colloidal gold particle labeling within the cytoplasm (Bode et al., 1993).Furthermore, when the two forms of H. pylori (spiral and coccoid) were compared using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH)-based proteomic, it was observed that the abundances of DNA gyrase subunits GyrA and GyrB were even higher in coccoid cells compared to their counterpart, suggesting active DNA synthesis in this population (Loke et al., 2016).
At the metabolic level, the coccoid form of H. pylori was found to maintain a basal level of activity reminiscent of bacterial cells in dormancy.Using bioluminescence assays, it was observed that the ATP levels in 9-to 13-day-old coccoid cells were much lower than that of bacilli although there was no indication of leakiness of this substrate into the extracellular space.Strikingly, ATP levels were found to slightly increased when fresh medium was added to the culture containing coccoid cells.This was suggested to be the preserved basal level of metabolic activity in coccoid H. pylori which could return to normal under optimal growth conditions.Nonetheless, the author recognized the limitation of the study as a single population of coccoid H. pylori (resulted from prolonged incubation) was analyzed and that those induced by different means such as antibiotic treatments could possess different biological profiles.They hypothesized that there existed two distinct coccoid sub-populations: the viable dormant form and the degenerative form, depending on the mode of induction (Sörberg et al., 1996).This notion is not uncommon since other studies had also reported observation of distinct coccoid populations (Bode et al., 1993;Willén et al., 2000).

| Degeneration
It is now known that coccoid formation in H. pylori can be induced by many conditions including but not limited to starvation, temperature fluctuations, changes in oxygen level, and antibiotic treatments.These conditions have been reported to have effects on the morphological transformation rates but were said to give rise to the same sequence of ultrastructural changes inside the cells.This led to postulations that all coccoid cells of H. pylori possess comparable structural and physiological properties despite the conditions used to induce them and that they are the manifestation of cell death (Kusters et al., 1997).
Cellular and genetic contents of the coccoid form were compared with those of bacilli as well as between coccoid populations induced by different conditions.At the nucleic acid level, DNA and RNA contents were found to have reduced drastically in all coccoid populations with no detectable DNA and almost no RNA detected upon coccoid induction by kanamycin.This observation was hypothesized to be due to degradation and fragmentation of the genetic materials during the morphological transformation leading to cell death.Although this trend was observed for all conditions, coccoid forms induced by prolonged incubation in microaerobic environment were able to maintain the highest level of both DNA and RNA contents, markedly higher than those of antibiotic-treated populations (Kusters et al., 1997;Narikawa et al., 1997).
To further investigate their viability, coccoid were assessed using the potential-sensitive probe DiOC 5 -3 and it was observed that only the background-stained population was present in an aged culture, suggesting loss of membrane potential (Kusters et al., 1997).
However, this observation was contradicted by a separate study demonstrating retention of rhodamine 123, a different cationic potential-sensitive dye, in coccoid H. pylori induced by prolonged incubation indicating the presence of membrane potential in this population (Sarafnejad et al., 2007).It is intriguing that the former study ruled out completely the likelihood of VBNC and argued that coccoid H. pylori induced in all conditions tested were completely degenerative while the data presented in the latter suggested some possibility of viable cells in the naturally aged population induced by prolonged incubation in microaerobic environment.Additionally, the ATP levels of coccoid populations were assessed and were found to have reduced drastically.While this suggested decline of cellular activities inside the cells, the author acknowledged, although with high skepticism, the possibility of coccoid cells maintaining the basal level of metabolic activity (Enroth et al., 1999).
Proteomic studies showed no difference in the total amount of protein per cell in the bacilli and coccoid form independently of conditions used to induce the latter (Kusters et al., 1997).Twodimensional polyacrylamide electrophoresis (2-D PAGE) data of H. pylori helical rods in exponential phase and those of 7-day-old coccoid cells in liquid culture revealed some 1500 proteins in both forms; however, only a few stage-specific protein species were identified.Coccoid-specific protein species were reported to belong to diverse classes including chaperones, ribosomal enzymes, and urease.Yet, they were said to be variants of proteins that were also abundant in rod-shaped cells, indicating post-translational modification and not de novo synthesis (Bumann et al., 2004).On the contrary, conflicting observations were made by a separate study which observed active de novo protein synthesis in the spiral and coccoid forms after induction of both populations by acid shock at pH 3.5 and 2.0, mimicking the human stomach environment.The protein patterns were observed to be different between the two forms, suggesting stage-specific proteins in the coccoid population.They were also hypothesized to belong to the heatshock protein family functioning as chaperones and enzymes involved in cytoskeletal rearrangement (Mizoguchi et al., 1998).The possibility of coccoid H. pylori remaining viable in acidic environment comes as no surprise since studies have shown that culturing of this specie could only be achieved in about one third of the gastric juice obtained from patience who tested positive for H. pylori by rapid urease test (RUT) and histology or by PCR-based method, suggesting the presence of coccoid form in the other samples collected (Yoshida et al., 1998;Young et al., 2000).

| A tale of two CTs (coccoid types)
Despite the stance different researchers take on the viability of the coccoid form of H. pylori, it is quite undeniable that there emerges a trend among the studies presented suggesting at least two types of coccoid cells that differ in size, genetic content, protein profile, membranous structure, metabolic activity, and presence of electron-dense bodies (Bode et al., 1993;Cellini et al., 2001;Kusters et al., 1997;Mizoguchi et al., 1998;Sörberg et al., 1996;Willén et al., 2000).While the coccoid types in most of these studies were observed to have resulted from the different conditions used to induce them, some other studies reported distinct coccoid types within the same population induced by a single condition (Cellini et al., 2001;Saito et al., 2003Saito et al., , 2008;;Sato et al., 2003).
Among the first to investigate coccoid types within a single population was Cellini et al. who showed the emergence of two types of coccoid cells during the transformation due to prolonged incubation.One sub-population was found to possess electron-dense bodies (EDBs) while the other did not and was thus suggested to be necrotic cells (Cellini et al., 2001).The EDBs observed were thought to represent apoptotic bodies containing DNA which was demonstrated in the study.This adds another layer to a prior hypothesis which suggested EDBs to be the energy reservoir in dormant coccoid cells since they were identified to consist of polyphosphates (Bode et al., 1993;Willén et al., 2000).Cellini and colleagues had a divergent perspective and believed there could be an apoptosis-like phenomenon that took place to establish an equilibrium between the different forms of H. pylori and this could be an act of altruism for species preservation (Cellini et al., 2001).
Two types of coccoid H. pylori, distinguishable by their membranous structures, had also been suggested through ultrastructural studies of the transformation using electron microscopy (Saito et al., 2003;Willén et al., 2000).Type A cocci were said to have an irregular or rugged surface and exhibited cell-to-cell contact behavior while Type B was described to possess a smooth surface with flagella wrapped around and showed no interaction with nearby cells.On top of having a rugged surface, Type A was surprisingly observed to display a discontinuous membrane and sometimes appeared to be made from multiple cells.Type B, on the other hand, possessed intact membranous structure which was suggested to be a sign of viability (Saito et al., 2003).In a follow-up study, the DNA content and CagA in Type A cocci were tracked using immunoelectron microscopy to assess the possibility of horizontal gene transfer within this sub-population.It was observed that some coccoid cells adhered to one another or even with a non-coccoid cell with the help of their flagella.Subsequently, the membrane boundaries between those cells became less distinctive followed by appearance of DNA and CagA in this particular area but none in the extracellular space.
These observations led them to believe that there existed a speciespreservation strategy between the two coccoid types where one underwent spontaneous autolysis and donated its DNA content to another (Saito et al., 2008).
Additionally, the two coccoid types have also been observed in other conditions and their abundances were reported to be condition-dependent.In one study, coccoid cells induced from 3-day aerobiosis were observed to be predominantly cells with a smooth surface similar to Type B coccoid.However, at Day 7 of aerobiosis most cells were found to have severely irregular shapes indicative of degradation with the presence of some spherical cells that exhibited rough surfaces similar to that of Type A. On the other hand, coccoid cells induced from prolonged incubation in microaerobic environment were found to be mostly Type B at Day 3 and then predominantly Type A at day 20 with few degraded forms observed (Mizoguchi et al., 1998).In another study, the addition of 300 mM glucose to Brucella broth supplemented with 10% heat-inactivated horse serum induced mostly Type B coccoid and retained a surprisingly high percentage of spiral forms while maintaining culturability (Saito et al., 2003).In vivo, Wen et al. examined the gastric mucosa of human patients obtained through endoscopic biopsy after 6 weeks of antibiotic treatment and found the presence of coccoid cells which they described to be round or irregular in shape, had thick cell walls, and reduced periplasmic space (Wen et al., 1999).These coccoid cells most likely resemble Type B as they possess a firm and well-defined cell wall with the presence of electron-dense granules.

| CELL S HAPE MECHANIS MS IN BAC TERIA
While searching for definitive proof pertaining to the viability of coccoid H. pylori is a challenging task, demonstrating the molecular mechanism of its formation has been shown to be just as difficult, if not more.In fact, this is not unique to H. pylori.The formation mechanism of VBNC in other bacteria has not been elucidated either and only two major hypotheses have been proposed so far.The "Cell Recession Theory" postulates that the VBNC form results from oxidative stress damage and growth inhibition imposed by stress conditions whereas the "Gene Regulation Theory" attributes this phenomenon to dynamic gene regulation (Dong et al., 2020).
The peptidoglycan layer encases bacteria to withstand turgor pressure and thereby preserving their cellular integrity.It has become more and more evident that the morphological transition during VNBC formation, such that in H. pylori, is preceded by substantial modification in the peptidoglycan or cell wall (Chaput et al., 2006;Costa et al., 1999).Thus, regardless of which pathway a bacterial cell may take to enter the VBNC state, it is very likely that it implicates the peptidoglycan biosynthesis and remodeling.

| Peptidoglycan biosynthetic pathway
The PGN biosynthetic pathway is complex and dynamic whereby it involves many proteins and interactions between them.This process begins in the cytoplasm where the precursor lipid II is synthesized.TPase (Egan et al., 2020).Turnover of peptidoglycan as well as generation of bacterial cell shape both require active involvement of hydrolases.A large number of PGN hydrolases have been discovered in different bacterial species and major classes of these enzymes include N-acetylmuraminidases (lysozymes and lytic transglycosylases), N-acetylglucosaminidases, amidases, endopeptidases, and carboxypeptidases (Egan et al., 2020).
Given the complexity of the PGN structure and biosynthetic pathways, most of its machineries require precise coordination and interaction to properly function.Two main PGN machinery complexes have been identified in bacteria-the elongasome and the divisome (Szwedziak & Löwe, 2013).While most bacteria rely on both complexes for growth and division, spherical bacteria utilize only the latter (Den Blaauwen et al., 2008).Scaffold proteins and cytoskeletal components all play important roles in providing spatial-temporal coordination and structural support for both machinery complexes.
In most rod-shaped bacteria, the cytoskeletal component MreB has been identified as a key player of the elongasome and aids in directing the insertion of nascent PGN during cell growth (Figge et al., 2004;Ursell et al., 2014).FtsZ is a homolog of the eukaryotic protein tubulin.This cytoskeletal protein polymerizes into a ring during cell division and recruits other components of the divisome to the division site (Adams & Errington, 2009;Bi & Lutkenhaus, 1991;Löwe & Amos, 1998).Both MreB and FtsZ are essential in many bacterial species and contribute substantially to their cell shape.For instance, bacteria have been observed to lose its cylindrical rod shape when MreB is depleted or disturbed and to filament in ftsZ mutants (Feucht & Errington, 2005).

| CELL S HAPE MECHANIS MS IN H . pylo ri
The PGN assembly machinery, PGN modifying enzymes (Figure 1) as well as non-enzymatic proteins (Figure 2) are all crucial to cell spiral shape generation and maintenance in H. pylori (Figure 2a).Only three PBPs are present in this bacterial species which are PBP1, PBP2, and PBP3 (Alm et al., 1999).PBP1 belongs to class A and has been demonstrated to induce spherical cells in H. pylori when depleted (Boneca et al., 2008).PBP2 is a TPase that interacts with the scaffolding protein MreC to form the core of the elongasome complex in this organism.Depletion of either of these proteins or inhibition of their interaction also lead to spherical cells (Figure 2d) (Contreras-Martel et al., 2017;El Ghachi et al., 2011).Lastly, the transpeptidase activity of PBP3 is known to be essential to cell division in this species since its depletion leads to filamentation (Figure 2f) (Boneca et al., 2008).
PGN modifying enzymes that have been identified in H. pylori so far include lytic transglycosylases, amidases, and peptidases (Figure 1).Slt and MltD are lytic transglycosylases capable of hydrolyzing the β-1 → 4 glycosidic bond which links N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) of the peptidoglycan in this organism (Chaput et al., 2007).N-acetylmuramoyl-L-alanyl amidase (AmiA) removes the peptide stem (L-alanine) from the glycan chain at the NAM moiety.This enzyme plays crucial roles in peptidoglycan metabolism, daughter cell separation, virulence, and morphogenesis in H. pylori.Its mutant was observed to have a chaining phenotype and fail in morphological transition (Chaput et al., 2006(Chaput et al., , 2016)).
Csd6 is an L,D-CPase capable of trimming GM4 into tripeptide (GM3) monomers and Csd4 is a D,L-CPase of the M14 family responsible for trimming GM3 further into dipeptide (GM2) monomers (Chan et al., 2015;Kim et al., 2014Kim et al., , 2015)).Inactivation of either gene generates straight rod cells in this organism (Figure 2c) (Sycuro et al., 2012).Fascinatingly, bioinformatics analysis of Csd6 revealed an L,D-TPase catalytic domain known as YkuD which can possibly catalyze the 3-3 crosslinking of peptidoglycan and has been identified in a range of bacteria (Biarrotte-Sorin et al., 2006;Frirdich et al., 2014;Kim et al., 2014Kim et al., , 2015)).However, this protein exhibits no L,D-TPase activity consistent with absence of 3-3 crosslinks in H. pylori, and instead functions as an L,D-CPase due to its unique conformation of the active site pocket which closely resembles that of typical carboxypeptidases rather than transpeptidases (Kim et al., 2015).Spatial-temporal coordination of both PGN assembly and remodeling by scaffold proteins and cytoskeletal components has been demonstrated to be just as crucial to cell shape generation and maintenance in H. pylori.CcmA is a bactofilin capable of filamentation and has been shown to interact directly with Csd7 and Csd5; both are non-enzymatic transmembrane scaffold proteins suggested to provide structural support separately for the Csd1-Csd2 complex and for the PGN precursor synthase, MurF (Salama, 2020).The actin homolog MreB is present in H. pylori but was initially thought to play no essential roles in cell shape regulation in this species (Waidner et al., 2009).However, new evidence has emerged and challenged this initial finding, suggesting that mreB is essential in H. pylori and this protein is crucial to generation of the helical shape in this organism (Taylor et al., 2020).

| Determinants of helical shape in H. pylori
When investigating the helical shape of H. pylori, Taylor et al. observed that these cells had maintained areas of positive and negative Gaussian curvatures.The positive Gaussian curvatures are the convexes along the cell body that have proportionately higher sidewall area.The negative Gaussian curvatures are the concaves found on the other side of the cell marked by lower proportion of sidewall area.The helicity was suggested to conform to a unique pattern created by two axes running along these curvatures.The major axis is found along the positive Gaussian curvature (convex) and the minor axis is on the negative Gaussian curvature (concave) (Figure 3).

Enrichment of PGN synthesis has been reported along the two axes of H. pylori with trends that highly correlate with localization of
CcmA and MreB respectively (Sichel et al., 2022;Taylor et al., 2020).
The current working model postulates that H. pylori grows by default as straight rod through MreB coordination, and the helicity is then generated by addition of the major axis along the lateral body through the coordination of CcmA.Yet, this alone is insufficient for maintenance of this peculiar shape.
The PGN hydrolases such as Csd1-Csd2, Csd3/HdpA, Csd4, and Csd6, have all been hypothesized to contribute to the generation of the helical shape of H. pylori through PGN relaxation.
However, their precise role and dynamics in the process are poorly Campylobacter jejuni, an ε-proteobacterium.Analysis of these gene sequences suggested their unique presence in curved-rod bacterial species (Caccamo & Brun, 2018;Esson et al., 2016).Csd6 and Pgp2 both possess a domain called Nuclear Transport Factor 2 (NTF2).This domain has recently been described to bind directly to the peptidoglycan and is responsible for convex cell-face localization of an L,D-carboxypeptidase in Bdellovibrio bacteriovorus (Banks et al., 2022;Lin et al., 2021).It is therefore highly probable that Csd4 and Csd6 are involved in generating convexes and curves in H. pylori.Nevertheless, it is still unclear how alterations in PGN monomers contribute to the overall cellular curvature (Chan et al., 2015;Kim et al., 2015;Sycuro et al., 2012).One fascinating observation in csd4-knockout mutants was a dramatic increase in GM4-GM3 dimer proportion compared to that of the wildtype (Sycuro et al., 2012).
Conceivably then, these enzymes indirectly contribute to a further reduction of the PGN dimers through limiting the availability of the substrates required for this activity.The question that remains is whether Csd3/HpdA, the only other D,D-endopeptidase in H. pylori, facilitates insertion of nascent PGN at the negative Gaussian curvatures where MreB is found to be concentrated.Since Csd3/ HdpA possesses the two enzymatic activities of D,D-endopeptidase and D,D-carboxypeptidase, perhaps regulation of its carboxypeptidase activity could give rise to a marker distinguishing the negative Gaussian curvature from its counterpart.

| Determinants of coccoid shape in H. pylori
In examining the mechanism of coccoid formation in H. pylori, at least two scenarios could be considered.In the first scenario, this phenomenon depends solely on substantial relaxation in the PGN network which eventually leads to a spherical shape as dictated by the turgor pressure.In this case, a spherical cell with the two cellular poles positioned opposite from each other should be observed (Figure 4A).However, this would only be plausible if PGN hydrolysis takes place without bias on both sides of the lateral body.On the other hand, PGN relaxation biased to a single side of the lateral body would result in a bleb also due to the turgor pressure.Expansion of this bleb would then push the two cellular poles closer to each other creating a coccoid cell with the two poles on the same side (Figure 4A).
In the second scenario, the coccoid formation involves both PGN relaxation and de novo PGN synthesis to generate the overall coccoid phenotype.In this case, the position of the two poles is dependent upon the precise localization of the two cellular activities.In fact, it has been observed that H. pylori goes through an intermediate curved or U shape, in which the two poles are close to one another, before eventually changing completely into a spherical cell when undergoes coccoid formation (Kusters et al., 1997;Willén et al., 2000).This suggests a dynamic process in both the PGN assembly and remodeling pathway, reminiscent of the second scenario (Figure 4B).
Mutants of H. pylori lacking either csd1, csd2, csd4, or csd6 have all been shown to be capable of transforming into coccoid under prolonged incubation (Sycuro et al., 2010).Thus, the potential roles of these PGN hydrolases in coccoid formation of this specie could be very minimal, if any, despite their importance in generation of cellular curvatures and twists.However, it is now becoming more evident that not all coccoid cells of H. pylori are the same.Thus, the coccoid forms induced from knockout mutants of these genes could have different properties from those induced from wildtype, especially in their PGN architectures and profiles.∆csd3/hdpA on the other hand was found to severely delay the transformation process in some strains while abolishing it altogether in others (Bonis et al., 2010;Sycuro et al., 2010).Furthermore, overproduction of this protein was shown to accelerate the morphological transition (Bonis et al., 2010).These indicate that the coccoid formation in H. pylori might depend on at least the activity of HdpA to generate GM-tetrapeptide (GM4) monomers.Fascinatingly, analysis of the PGN profile of some coccoid populations revealed accumulation F I G U R E 3 Generation of helical shape in Helicobacter pylori.The helical shape of H. pylori conforms to the pattern of a major helical axis (pale red line) and a minor helical axis (pale yellow line) that extend along its lateral body.Accumulation of CcmA filaments (red line) has been reported to follow the major helical axis and coincide with relative increased in peptidoglycan synthesis, contributing to generation of convex regions.MreB has also been observed to localized in zones along the minor helical axis and coincide with relative increased in peptidoglycan synthesis, contributing to formation of concave regions.
of GM-dipeptide (GM2) monomers, a product two-step downstream from the GM4 (Chaput et al., 2006;Costa et al., 1999;El Mortaji et al., 2020).However, it is still not known whether this muropeptide plays an active role in the transformation or if it is just a by-product resulting from the cleavage of upstream muropeptides.So far, current findings support the latter speculation since this bacterium has already been shown to become coccoid in late stationary phase in the absence of Csd6 and Csd4, the enzymes responsible for trimming GM4 into GM3 and GM3 into GM2, respectively (Sycuro et al., 2010).Perhaps there exists a distinct mechanism of coccoid formation which is independent of the activity of these PGN carboxypeptidases.Nonetheless, this unique PGN profile has been demonstrated to confer benefits in H. pylori by aiding in its evasion of immune recognition (Chaput et al., 2006).Altogether, these show that investigations on the PGN hydrolases such as Csd3/HdpA are needed to help further our understanding of coccoid formation in this organism.
Localized enrichment of PGN synthesis and alteration of PGN crosslinks are both central to the bends and twists of H. pylori (Sycuro et al., 2010;Taylor et al., 2020).It is possible that some of the same machineries involved in generation and maintenance of the spiral form are involved in coccoid formation.MreB has been shown to preferentially localize at the negative Gaussian curvatures of H. pylori and has been suggested to play an essential role in its helical shape (Taylor et al., 2020).It is somehow counterintuitive that this cytoskeletal protein plays a role in coccoid formation since spherical form is deprived of concaves and cell elongation does not seem to be at play in this process.However, if MreB does indeed interact with the PBP2:MreC complex in H. pylori, then disruption of its localization could possibly sabotage the regulation of the elongasome, potentially leading to uncoordinated activity of this complex and eventually resulting the in loss of lateral growth and maintenance of the cylindrical shape.Likewise, it has been shown that a H. pylori mutant of ccmA possesses a curved morphology as cells lose the ability to create twists (Sycuro et al., 2010).Thus, it is also likely that disruption of CcmA localization or filamentation could synergistically abolish the helical-cylindrical morphology of this organism when its partner complexes such as Csd1-Csd2:Csd7 and Csd5:MurF become unregulated.
Accordingly, if coccoid formation in H. pylori indeed requires de novo PGN synthesis, then components of the divisome such as PBP3 could possibly be at play.However, this notion is also counterintuitive since coccoid cells of H. pylori are hypothesized to be dormant and have not been observed to be able to divide.Nonetheless, this cannot rule out completely the potential role of this protein in the process, especially since this organism possesses a minimal set of genes involved in the PGN pathways (Figure 1).Perhaps there exists a unique mechanism in which PBP3 mediates the switch from cell division to coccoid formation when cell elongation machineries fail to mobilize.Class A PBP1 is another enzyme likely involved in de novo PGN synthesis during coccoid formation.It has been shown to facilitate both cell elongation and cell division in H. pylori as well as playing a critical role in PGN repair (Typas et al., 2012).However, current knowledge on its activity in other bacterial species suggests that this protein contributes minimally to cell shape but bears an essential role in maintenance of the cell wall integrity (Vigouroux et al., 2020).All in all, further investigations on the dynamics of all three PBPs during the morphological transformation could shine light on whether de novo PGN synthesis is essential to coccoid formation in H. pylori.

| Type I toxin-antitoxin system in H. pylori
As the name implies, toxin-antitoxin (TA) is a two-component system made up of a toxin and an antitoxin protein or RNA that are agonistic to one another.They are small genetic elements ubiquitously found in bacterial genomes and have been shown to possess many roles ranging from plasmid stabilization to stress responses and persistence (Jurėnas et al., 2022).Based on the mechanism in which the antitoxin naturally takes to interact with its counterpart, TA systems can be divided into 7 classes.Type I TA system encodes for a toxin RNA and an antitoxin antisense RNA on the opposite strand in the same locus.When expressed, the toxin RNA gets processed into an active mRNA and is subsequently translated into a short peptide.
Overexpression of this peptide has been associated with growth arrest and morphological transformation (El Mortaji et al., 2020;Peltier et al., 2020).In normal physiological condition, the activity of this toxin is regulated by the antitoxin antisense RNA.This unstable but abundant antisense RNA can interact with the toxin RNA to form a duplex, which then serves as a marker for degradation by RNase (El Mortaji et al., 2020;Masachis & Darfeuille, 2018).Consequently, the production of the toxin peptide is highly regulated at the RNA level and an imbalance between the two components leads to cellular toxicity.
A family of six type I TA systems has been identified in the chromosome of H. pylori strain 26695 and has been shown to be conserved in over 60 strains of this species.Each of these systems encodes for a short peptide designated Antisense-associated peptide family A (AapA) and an antisense RNA (IsoA), except for aapA2 due to lack of a functional promoter.Transcriptomic study revealed that they are constitutively expressed in vivo during exponential phase and translation of the mRNAs was demonstrated to be inhibited by their respective cognate antisense RNA (Arnion et al., 2017;Sharma et al., 2010).When AapA1 was overproduced in strain B128 of H. pylori, cells were found to rapidly transform into cocci.Interestingly, the same study also observed that oxidative stress induced by H 2 O 2 could create an imbalanced expression of this system leading to the same coccoid phenotype.Biochemical analyses on these cells revealed that they had intact membrane integrity, reduced ATP content, disturbed membrane potential, and an accumulation of GM2, a putative marker of coccoid H. pylori, and were thus suggested to be the VBNC form (El Mortaji et al., 2020).

| Putative mechanism of coccid formation induced by AapA1
AapA1 toxin has been suggested to interfere with cell elongation and cell division in H. pylori, leading to the coccoid phenotype observed in this organism (Bendezú & de Boer, 2008).This speculation came as no surprise since the interaction of a type-IV toxin in E. coli, CbtA, with MreB and FtsZ has been shown to influence the morphology of this organism (Heller et al., 2017;Taylor et al., 2020).E. coli cells were observed to become lemonshaped when both cell elongation and division were disrupted but became spherical when only cell elongation was inhibited (Heller et al., 2017).Interestingly, it has also been observed that spherical or coccoid E. coli cells with defects in cell elongation did not undergo lysis and could propagate as spherical cells when the growth rate is reduced (Bendezú & de Boer, 2008).Although AapA1 is a smaller peptide compared to CbtA type-IV toxin in E. coli, this does not rule out the possibility that this toxin can influence the morphology of H. pylori, especially since this organism has been shown to have a unique cell-shape mechanism owing to its minimal set of genes involved in PGN biosynthesis.Therefore, we propose a novel mechanism of coccoid formation in H. pylori through induction of AapA1 type-I toxin.We reason that the coccoid cells induced by this mechanism are similar to those induced by prolonged incubation and are viable due to the intrinsically slow growth rate of this species.
When there is an imbalance between the Aapa1 toxin and the IsoA1 antitoxin RNAs, the toxin mRNA no longer gets targeted for degradation by RNase and can be translated into short alphahelix peptides.In vivo and in silico structural studies indicate that AapA1 peptides localize in the inner membrane of H. pylori and are capable of inserting itself into the lipid bilayer (El Mortaji et al., 2020;Korkut et al., 2020).An interaction between the phospholipid head groups and the positively charged C-terminus of the toxin has been predicted, as well as a possible interaction between the peptides themselves through their hydrophobic regions (Korkut et al., 2020).Although it is still not apparent whether these alpha helices can assemble into pores like other type-I toxin peptides, this phenomenon is unlikely since the coccoid cells induced by overexpression of AapA1 toxin has already been shown to have no ATP leakage.Thus, the integrity of the membrane is intact; however, disruption of the membrane potential was reported (El Mortaji et al., 2020).
In H. pylori, MreB is a membrane-associated protein that transiently localizes to negative Gaussian curvatures during growth.Thus, it is likely that its interaction with the inner membrane is relatively weak to allow for easy mobilization as it regulates the elongasome complex at this site.Therefore, membrane potential could possibly play a central role in this interaction and its disruption could result in failure of MreB to adhere to the inner membrane.In fact, this has already been demonstrated in at least two other bacterial species including Bacillus subtilis and Caulobacter crescentus (Strahl & Hamoen, 2010).At the same time, MreB activity is known to be ATP-dependent whereas ATP has been shown to be depleted in the coccoid form of H. pylori (Dye et al., 2011;El Mortaji et al., 2020;Enroth et al., 1999;Sörberg et al., 1996).
Consequently, the activity of this protein could be reduced drastically when the coccoid formation is initiated which would subsequently lead to unregulated synthesis of PGN by the PBP2:MreC elongasome.Meanwhile, disruption of the membrane potential could also affect the activity of CcmA and in turn Csd1-Csd2:Csd7 as well as Csd5-MurF complexes along the major helical axis, diminishing cellular helicity altogether.From the intermediate form, H. pylori has been hypothesized to go into a complete coccoid through outward extension of the cell wall at the concaved negative Gaussian curvature.This could be the effect of PGN relaxation at this site and is likely facilitated by Csd6, Csd4, and especially Csd3/HdpA since overexpression of this protein leads to accelerated coccoid formation; this ultimately results in accumulation of GM2 as observed in some coccoid population analyzed (Bonis et al., 2010;Sycuro et al., 2010Sycuro et al., , 2012)).
It is still not known whether MreB of H. pylori directly regulates the elongasome the same way as has been observed in other species such as C. crescentus or if a third partner is required for this interaction (Divakaruni et al., 2007;Li & Gao, 2021).Current knowledge of cellshape regulation in H. pylori strongly suggests that this species has a unique mechanism for generating and maintaining its shape, especially its helical shape.Thus, further investigation into the relationship of these proteins is needed since this might be the key to understanding the coccoid formation in this organism.Furthermore, it is also not known whether the membrane potential gets disrupted during the morphological transformation in late stationary phase.Perhaps the accumulation of toxic byproducts or changes in pH inside the medium resulting from prolonged incubation could also induce the same TA system (AapA1/IsoA1) through p(p)Gpp which eventually leads to the same coccoid formation mechanism.All in all, further studies are needed to investigate the possibility of the hypothesis proposed as well as to search for other stress conditions that could potentially activate this TA system in H. pylori leading to its coccoid formation.
Lipid II gets transported or flipped into the periplasm by proteins called flippases and is then utilized by PGN assembly machineries.Two important classes of enzymes involved in the subsequent steps are glycosyltransferases (GTases) and transpeptidases (TPases), which catalyze glycan chain polymerization and peptide crosslinking respectively (van Teeseling et al., 2017).Penicillin-binding proteins (PBPs) of class A consist of both GTase and TPase domains and can thus catalyze both aforementioned reactions.Class B PBPs, on the other hand, are monofunctional enzymes and consist of only understood.Although it is quite plausible that the endopeptidase activity of Csd1-Csd2 facilitates the increased insertion rate of nascent PGN subunits at the positive Gaussian curvatures where CcmA localizes, this warrants further investigation.Csd4 and Csd6 of H. pylori are respective homologs of Pgp1 and Pgp2 proteins in F I G U R E 2 Morphological variants of Helicobacter pylori as implicated by the cell shape determinants.(a) Cartoons depicting the helicalrod shape of wildtype H. pylori.(b) A curved-rod morphology is observed in the mutants of Δcsd1, Δcsd2, Δcsd5, Δcsd7 and ΔccmA.(c) Δcsd4 and Δcsd6 mutants generate a straight-rod morphology.(d) Depletion of either PBP1, PBP2, MreC or overexpression of HdpA all lead to spherical shape.(e) Δcsd3 knockout generates an irregular, branched morphology, or a curved rod.(f) PBP3 depletion results in long filamented morphology.

F
Generation of coccoid form in Helicobacter pylori.Cartoon representation describing two possible scenarios of morphological transformation in H. pylori.(A) The first scenario relies solely on extensive relaxation in the peptidoglycan layer.If hydrolysis occurs without bias (top), the PGN gets heavily modified on both sides of the lateral body (a), then the turgor pressure from inside the cell pushes against the relaxed cell wall (b) resulting in loss of shape and becomes spherical with two cellular poles on the opposite sides of each other (c).In the case that PGN relaxation occurs preferentially to a single side of the lateral body (bottom), a bleb or a bulge would form at the site where hydrolysis localizes (b) and would continue to expand and push the two poles close to each other (c) eventually leads to a round cell with the two poles on the same side (d).(B) In the second scenario, both relaxation and de novo synthesis of the peptidoglycan are required.From the helical-rod form (a), uncoordinated insertion of nascent PGN transforms the cell into a U shape (b) bringing the two cellular poles close to each other.(c) Peptidoglycan relaxation transforms the cell further into a spherical form due to the turgor pressure.Finally, a spherical coccoid cell is formed with the two cellular poles on the same side, close to each other (d).