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Inhibition of Type IV Secretion Systems in Pathogenic Bacteria


By: Carol A. Rouzer, VICB Communications
Published:  May 20, 2016



Peptidomimetic small molecules block effector translocation associated with poor outcomes in Helicobacter pylori infection..


Bacteria produce many kinds of effector molecules that enable them to survive in a hostile environment. For bacteria living in close contact with other cell types, the activity of an effector molecule may depend on its translocation into the neighboring cells through the action of specialized target delivery systems. One such delivery system is the type IV secretion system (T4SS), a family of contact-dependent transport machines characterized by its versatility and diversity. Found in a broad range of bacterial species, T4SSs comprise a complex of conserved core subunits and species-specific subunits. They can be broadly classified on the basis of one of three functions: DNA conjugation, DNA uptake/release into the extracellular environment, or effector translocation. In a range of pathogenic bacteria, T4SSs enable evasion of host defense mechanisms and/or alteration of normal host cell metabolic functions. Thus, T4SSs are virulence factors for some important bacterial infections. This led Vanderbilt Institute of Chemical Biology member Maria Hadjifrangiskou and her laboratory to search for small molecule inhibitors of T4SS function. They report the results of initial studies identifying two compounds that have activity across multiple T4SSs and bacterial species [C. L. Shaffer, et al., mBio., 7, e00221-16].


Helicobacter pylori is a frequent cause of persistent infection in the human gastric mucosa. Although asymptomatic in most people, H. pylori infection can cause serious illness, including gastritis, ulcers, and cancer. Poor outcomes are more likely if the infecting bacteria express the cag T4SS, an effector translocation system encoded by the cag pathogenicity island (cag PAI). The cag T4SS mediates the translocation of the CagA oncogenic protein and peptidoglycan directly into gastric epithelial cells through pili, filamentous structures that form at the interface between the bacterial and host cells. CagA protein is present at the tips of these pili, and their presence is required for CagA translocation. The association between T4SS expression and virulence in H. pylori led the Hadjifrangiskou lab to hypothesize that inhibitors of T4SS could prevent poor outcomes in H. pylori-infected patients. Prior work had identified a series of peptidomimetic 2-pyridones (Figure 1) that block pilus formation via the chaperone-usher pathway in E. coli, suggesting that compounds of similar structure might block cag T4SS-dependent pilus formation as well. Thus, the investigators created a library of 22 compounds based on the 2-pyridone scaffold. They screened this library by monitoring Cag4-mediated responses in gastric epithelial cells, including activation of NF-κB signaling and secretion of interleukin-8 (IL-8) (Figure 2). The screen identified two compounds designated C10 and KSK85 (Figure 1) that inhibited these responses in H. pylori-exposed gastric epithelial cells in a dose-dependent fashion. The effects of the two compounds were additive, suggesting that they work via distinct mechanisms. A third compound, GKP42, served as an inactive control for further studies.



FIGURE 1.Structures of the peptidomimetic 2-pyridone scaffold (upper left) and the three compounds investigated in this research. Figure reproduced under the Creative Commons Attribution 4.0 International License from C. L. Shaffer, et al., mBio., 7, e00221-16. Copyright 2016, C. L. Shaffer, et al.




FIGURE 2. Scheme showing experimental design. A library constructed from compounds based on a peptidomimetic 2-pyridone scaffold was screened in search of molecules that block T4SS-mediated cellular functions, including activation of NF-κB signaling, induction of IL-8 secretion, and the translocation and phosphorylation of CagA protein. Active compounds were subjected to further experimentation to define structure-activity relationships and determine mechanism of action. Figure reproduced under the Creative Commons Attribution 4.0 International License from C. L. Shaffer, et al., mBio., 7, e00221-16. Copyright 2016, C. L. Shaffer, et al.



To verify that C10 and KSK85 block cag T4SS-mediated effector translocation, the investigators exploited the fact that CagA is rapidly tyrosine phosphorylated following its translocation into gastric epithelial cells. This enabled them to monitor CagA translocation by the appearance of the phosphorylated protein on immunoblot. The results confirmed that both C10 and KSK85 reduced CagA translocation in H. pylori-exposed gastric epithelial cells. In contrast, they did not impede release of VacA into the medium, a process carried out by a type V secretion system in H. pylori. Further studies demonstrated that the T4SS inhibitory effects of C10 and KSK85 were not due to toxicity in either the bacteria or the gastric cells.


Field-emission scanning electron microscopy showed that C10 and KSK85 did not prevent association between H. pylori and their target cells. However, KSK85 prevented pilus formation between the two cell types (Figure 3). This was not observed for C10 or GKP42, providing additional evidence that C10 and KSK85 inhibit T4SS-dependent translocation by different mechanisms.




FIGURE 3. Effect of vehicle (A), GPK42 (B), C10 (C), and KSK85 (D) on the formation of pili between H. pylori and gastric epithelial cells as seen by field-emission scanning electron microscopy. Arrows highlight the positions of pili, which can be seen in all micrographs except for (D). Figure reproduced under the Creative Commons Attribution 4.0 International License from C. L. Shaffer, et al., mBio., 7, e00221-16. Copyright 2016, C. L. Shaffer, et al.



Cag T4SS-mediated activation of NF-κB and secretion of IL-8 can result from translocation of either CagA or peptidoglycan. The investigators found that the response of gastric epithelial cells to C10 was the same whether the cells were exposed to wild-type H. pylori, or a ΔcagA H. pylori mutant. These findings suggested that CagA translocation is not needed to observe the suppressive effects of C10, implying that peptidoglycan was the primary mediator of NF-κB activation and IL-8 secretion in this case. An unexpected finding was that the effects of KSK85 were actually increased in gastric cells exposed to the ΔcagA H. pylori mutant as compared to the wild-type bacteria. The investigators hypothesized that an interaction between the CagA protein and KSK85 might partially block the compound’s activity in wild-type bacteria.


H. pylori uses the cag T4SS to modulate gastric epithelial cell polarity, thereby creating a favorable environment for its own survival. To investigate the importance of this function, the investigators monitored the growth of wild-type H. pylori and a ΔcagE mutant lacking a cag T4SS ATPase that is required for pilus formation. The mutant H. pylori adhered to gastric cells normally, but did not form pili. Fewer mutant than wild-type bacteria were recovered following a 6 h co-culture with gastric cells, suggesting a beneficial effect of the cag T4SS to H. pylori growth and survival. Addition of C10 or KSK85 had no effect on the growth of the ΔcagE H. pylori mutant, consistent with the absence of a functional cag T4SS in these cells. In contrast, although ΔcagA H. pylori grew at a rate comparable to that of wild-type cells in the absence of C10 or KSK85, addition of either compound substantially reduced growth of the cells. The compounds had no effect on the growth of wild-type bacteria. These results suggest that CagA helps the bacteria to overcome the effects of C10 and KSK85, possibly by promoting a more favorable environment for survival.


T4SSs are found in many species of bacteria, leading the investigators to explore the ability of C10 and KSK85 to block the activity of systems other than the H. pylori cag T4SS. Both compounds reduced the DNA conjugation efficiency of the IncN group conjugative T4SS encoded by pKM101 in E. coli. However, only C10 was able to reduce the activity of the IncF group R1-16 conjugative T4SS.


The vir T4SS of Agrobacterium tumefaciens has served as a prototype for the study of these secretion systems. The A. tumefaciens T4SS mediates translocation of DNA into recipient plant cells. To see if C10 or KS585 could block the vir T4SS, the researchers used a plasmid-derived transfer DNA (T-DNA) encoding β-glucuronidase. Infection of a tobacco leaf by bacteria carrying this plasmid enables vir T4SS-mediated transfer of the T-DNA into the plant cells, where it is incorporated into cellular DNA. Because the β-glucuronidase gene contains introns, it cannot be expressed by the bacteria. However, the eukaryotic plant cells can properly process the gene, leading to β-glucuronidase expression in regions of the leaf infected by the bacteria. The presence of the enzyme is revealed by histochemical staining (Figure 4), and can be quantified by a fluorescence-based assay. Using this assay, the researchers showed that C10 suppresses T-DNA transfer by the vir T4SS, whereas KSK85 had no effect. In a separate assay, the investigators also showed that C10 blocked vir T4SS-dependent tumor formation in carrots (Figure 5).



FIGURE 4. Assay for T4SS-dependent T-DNA translocation by A. tumefaciens. Bacterial strain GV3101 (upper circle) or GV3101 bearing a T-DNA plasmid encoding β-glucuronidase (lower circle) were infiltrated into leaves of the tobacco plant (Nicotiana benthamiana). Successful transfer of the T-DNA plasmid into plant cells enables expression of β-glucuronidase, which can be detected by the enzyme-mediated production of a blue-green dye. A fluorescence-based assay provided quantification of β-glucuronidase expression. Figure reproduced under the Creative Commons Attribution 4.0 International License from C. L. Shaffer, et al., mBio., 7, e00221-16. Copyright 2016, C. L. Shaffer, et al.




FIGURE 5. A. tumefaciens causes tumor formation in carrots via a vir T4SS-dependent mechanism, as seen in the case of the vehicle (DMSO)-treated disc above. C10, but not KSK85 or GKP42, suppressed this tumor formation. Figure reproduced under the Creative Commons Attribution 4.0 International License from C. L. Shaffer, et al., mBio., 7, e00221-16. Copyright 2016, C. L. Shaffer, et al.



The results demonstrate that two compounds, C10 and KSK85, can reduce the efficiency of cag T4SS-mediated effector translocation. Their effects are not limited to this system as C10, in particular, blocks the activity of multiple other T4SSs. It is interesting to note that the two compounds differ only by the presence of a methoxy group on KSK85 that is not present on C10 (Figure 1). Yet, KSK85 blocks cag T4SS-mediated pilus formation while C10 does not, and C10 blocks several T4SS-dependent processes that are not affected by KSK85. Clearly, C10 and KSK85 are valuable probes for the exploration of T4SS assembly and function. They also represent important hit molecules that can be the starting point for optimization efforts for the discovery of highly potent inhibitors of T4SSs. Based on the association of the cag T4SS with poor outcomes in H. pylori infection, such inhibitors could well be valuable assets for the treatment of these, and possibly other infectious diseases.



ViewmBio article: Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens










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