Why Study on Helicobacter pylori Type IV Secretion System is Slow?
KeywordsHelicobacter pylori Genomics Type four secretion system Research Slow pace
To The Editor,
Type IV secretion system (TFSS) is a diverse class of secretion system that has important roles in virulence of some gram-negative bacteria. Bacterial TFSS is a family largely comprised of translocation and conjugation machinery. Usually, it is present in gram-negative, gram-positive, and wall-less bacteria and is involved in the translocation of DNA and/or proteins across the cell envelope. The existence of TFSS in Helicobacter pylori was confirmed almost two decades ago and later demonstrated that it is involved in the delivery of CagA into gastric epithelial cells (associated with gastric carcinogenesis). The sub-units of H. pylori TFSS are encoded by a 40 kb cag PAI (cag Pathogenicity Island) region in the genomic DNA. The organization of the translated proteins into TFSS is poorly characterized but likely consists of a cytoplasmic part (mainly composed of ATPase), a middle part/core complex (that spans from the inner membrane to the outer membrane), and an outer membrane-associated part (mainly the pilus forming subunits). To illustrate the functional roles of the sub-units, individual ORFs of cag PAI were knocked out . Despite efforts from several research groups, the sub-unit structure, organization of TFSS, and its mechanism of action are poorly understood. This communication aims to explore the bottlenecks in H. pylori TFSS research. One confounding problem is the lack of sustained interest in the exploration of unanswered questions as evident by the lack of consecutive publications by any research group. Another major obstacle is the unavailability of a better understanding of the mechanism of H. pylori TFSS and lack of interest in the TFSS of bacterium itself. Here, we discuss some problems and difficulties encountered till now that have contributed to the slow pace of H. pylori TFSS research.
A Larger Number of Sub-Units in H. pylori TFSS
Unlike other organisms (Agrobacterium tumefaciens, Bordetella pertussis, Legionella pneumophila, Anaplasma phagocytophilia, and Ehrlichia chaffeensis), H. pylori TFSS is composed of several additional sub-units making it difficult to extrapolate functional roles from other systems. For example, Fischer et al.  attempted to elucidate the role of 27 gene products that form TFSS in translocation of CagA (an effector) and IL8 induction by systematic mutagenesis. Seventeen gene products were shown to have functional relevance in either cagA translocation or IL8 induction, while knock-out of the remaining did not show any obvious phenotype. The problem is compounded by the fact that most of the genes are unique to cag-TFSS, i.e., only present in cag PAI . Further, most of the genes are encoded for structural proteins with no enzymatic activity, thereby making it difficult to assign a role for this protein. Another strategy to determine their roles relies on having protein-protein interactions. The caveat in this strategy is the expression of membrane proteins in a soluble form utilizing a heterologous host. Proper folding cannot be ascertained by refolding of denatured proteins, discouraging the use of inclusion bodies. We should use diverse affinity tags and expression hosts to produce soluble recombinant protein before performing protein-protein interaction experiments .
Lack of Reproducibility in the Results
A major hurdle in Cag-TFSS research in H. pylori is inconsistency and lack of reproducibility of results. There are several instances where research groups have reported contradictory findings without adequate explanation for the discrepancy. Matters are further complicated when the use of similar systems, techniques, and tools results in diverse outcomes. The field is plagued with several such instances, for example, in 2007, Kwok et al.  reported that the RGD motif of cagL was essential for interaction with a host α integrin receptor. Moreover, in 2009, Jiménez-Soto et al.  showed this interaction is RGD motif independent. In another case, Fischer et al. (2001) reported  cagM isogenic null mutant displayed loss of cagT expression (Fig. 4 of this paper) but in 2008, Kutter et al.  showed cagM null mutant successfully express cagT (Fig. 4 and 6C of this paper). In 2003, Rhodes et al. 2003 , demonstrated that the presence of cag γ is essential for processing of cagY (Fig. 7A of this paper) but Kutter et al. in 2008  did not show any effect of cag γ on the processing of cagY (Fig. 4 of this paper). In another instance, Fischer et al. in 2001 , negated a role for cagF (Hp0543) and suggested a partial role of CagD in CagA translocation by measuring phosphorylated CagA protein in respective null strains . Interestingly, using the same method (measuring phosphorylated cagA) in null strains of cagF and cagD, different research groups have published contrasting results. For example, Cendron et al. (2009)  showed that there is complete abolishment of phosphorylated CagA in the null strain (Fig. 6 of this paper) supporting the Ghoula et al. (2004)  previous finding demonstrates the loss of phosphorylated CagA in CagF null strain. However, Couturier et al. in 2006  established a direct physical interaction between CagF and cagA and also suggested that cagF is essential for cagA localization. Thus, an inconsistency in published reports on the roles of individual sub-units is jeopardizing the pace of H. pylori TFSS research.
Use of Strains with Significant Variation in Gene Sequence
Genome sequencing of 43 H. pylori strains have been completed , and another bottleneck was identified that two individual strains may differ in as much as 13% of their gene content with up to 800 genes being strain specific . Thus, the use of different strains may account the wide range of discrepancies. However, a more worrisome trend is the repetition of published work using a different strain. Contradictory results are usually defended quoting differences in strain instead of visiting the experimental errors. This tendency has thwarted the forward progress in the field.
Lack of Proper Control and Proper Designing
Most of the researchers have determined the role of a gene/gene product merely by knocking out the gene and showing the loss of function [1, 8, 9]. Ideally, the gene’s role should be determined by showing loss of function (by knock out) as well as a gain of function due to complementation. Very few groups have used the complementation assay to conclude the role of the gene . Gene disruption studies in bacteria utilizing replacement of a gene with a marker gene can potentially result in polar effect, especially if that gene is a part of an operon. Thus, researchers are utilizing this strategy (knock-out) that should examine the polar effect by measuring the mRNA level and/or protein levels of the flanking gene product. For study of protein-membrane localization, we should not determine protein localization through the use of antibody raised against terminal peptide/small peptide from specific region because the region of protein against which the antibody was raised against may be buried in the membrane and another part which is exposed but antibody against that domain was not available .
Based on lessons from available literature and our own experience on TFSS, we would like to suggest strategies to decipher the roles of CagPAI encoded proteins. Research groups with access to all the isogenic null strain of CagPAI encoded genes, antibodies against all the proteins of cagPAI, and good shuttle vectors/system for complementation can test these strategies to decipher the H. pylori TFSS. The first strategy involves resolving H. pylori whole cell extract prepared under non-denaturing conditions through a native gradient PAGE followed by immunoblotting with subunit-specific antibodies . The subunit that forms the part of the complex will light up as a high molecular weight band and will be absent in an extract from an isogenic null strain, thereby clarifying the role in assembly of Cag-TFSS. The second strategy involves the expression of different gene combinations in CagPAI null strain along with the CagA gene. The combination that restores CagA translocation most likely forms the functional TFSS. Cryo-electron microscopy shall also be helpful in visualizing and deciphering the core-complex of H. pylori TFSS. Structure of core-complex of plasmid pKM101 encoded TFSS has been successfully illustrated by Fronzes et.al (2009) . Since H. pylori TFSS is composed of several proteins (unlike plasmid pKM101), interpretation of observation/data may be more cumbersome.
Authors are thankful to Uka Tarsadia University, Bardoli, (Gujarat), India, and the National Institute of Technology, Raipur (Chhattisgarh), India.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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