The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 ≤ 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 μM. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.
The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes. Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2 ؊/؊ mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2 ؊/؊ cells that the vacuole-to-cytosol transitioning of L. monocytogenes is greatly accelerated. Unexpectedly, we found that in Perforin-2 ؊/؊ macrophages, Listeria-containing vacuoles quickly (<15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that a L. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2 ؉/؉ and Perforin-2 ؊/؊ macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity. Both extracellular bacteria and virus-infected cells are targeted by innate defense responses that employ pore-forming proteins (1). Extracellular bacteria that become bound with the complement factor C3b and the C5b-8 complex trigger the polymerization of C9, resulting in a doughnut-shaped pore with a diameter of 100 Å that constitutes the membrane attack complex (MAC) (2-4). Similarly, virus-infected cells are recognized and eliminated by natural killer (NK) and cytotoxic T lymphocytes (CTL) that, as part of their respective killing programs, secrete Perforin-1, which forms a cluster of lethal pores in the membrane of the infected cell (5, 6). The complement proteins C6 to C9 and Perforin-1 all possess a membrane-attack-complex-perforin (MACPF) domain, which mediates the homopolymerization process that drives pore formation.A gene predicted to encode a third MACPF-containing protein, macrophage expressed gene-1 (Mpeg1), recently has been described in a number of invertebrates and zebrafish and plays a role in innate immune responses in these species to bacterial pathogens (7-11). Phylogenic analyses indicate that the MACPF domain of Mpeg1 is the ancestor of the MACPF domains in the complement and Perforin-1 proteins (12). Interestingly, although homologous Mpeg1 genes are found in most metazoan genomes spanning from sponges to humans, Mpeg1, or MACPF-encoding genes more generally, so far have not been identified in nonmetazoan clades of eukaryotes. However, the MACPF domain itself bears a striking structural si...
Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C. trachomatis replication. When bacterial replication was inhibited using KSK120, an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C. trachomatis inclusion expansion.
Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria . For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.
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