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ABSTRACT Shorter and more effective treatment regimens as well as new drugs are urgent priorities for reducing the immense global burden of tuberculosis (TB). As treatment of TB currently requires multiple antibiotics with diverse mechanisms of action, any new drug lead requires assessment of potential interactions with existing TB antibiotics. We previously described the discovery of wollamides, a new class of Streptomyces-derived cyclic hexapeptides with antimycobacterial activity. To further assess the value of the wollamide pharmacophore as an antimycobacterial lead, we determined wollamide interactions with first- and second-line TB antibiotics by determining fractional inhibitory combination index and zero interaction potency scores. In vitro two-way and multiway interaction analyses revealed that wollamide B1 synergizes with ethambutol, pretomanid, delamanid, and para-aminosalicylic acid in inhibiting the replication and promoting the killing of phylogenetically diverse clinical and reference strains of the Mycobacterium tuberculosis complex (MTBC). Wollamide B1 antimycobacterial activity was not compromised in multi- and extensively drug-resistant MTBC strains. Moreover, growth-inhibitory antimycobacterial activity of the combination of bedaquiline/pretomanid/linezolid was further enhanced by wollamide B1, and wollamide B1 did not compromise the antimycobacterial activity of the isoniazid/rifampicin/ethambutol combination. Collectively, these findings add new dimensions to the desirable characteristics of the wollamide pharmacophore as an antimycobacterial lead compound. IMPORTANCE Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur. Therefore, shorter, safer, more effective TB therapies are required, and these should ideally also be effective against drug-resistant strains of the bacteria that cause TB. This study shows that wollamide B1, a chemically optimized member of a new class of antibacterial compounds, inhibits the growth of drug-sensitive as well as multidrug-resistant Mycobacterium tuberculosis isolated from TB patients. In combination with TB antibiotics, wollamide B1 synergistically enhances the activity of several antibiotics, including complex drug combinations that are currently used for TB treatment. These new insights expand the catalogue of the desirable characteristics of wollamide B1 as an antimycobacterial lead compound that might inspire the development of improved TB treatments.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence. Author summary Listeria monocytogenes is a bacterial pathogen that causes severe, often fatal disease in vulnerable individuals. Listeria monocytogenes infects host cells, where it needs to escape from its entry vacuole to replicate inside the host cell cytoplasm and spread to neighboring cells. Inability of Listeria to escape from vacuoles has been associated with long-term bacterial survival inside infected immune cells and chronic infection. We discovered that inhibition of the master regulator of the bacterial factors that orchestrate the intracellular lifestyle of Listeria monocytogenes traps the bacteria inside their entry vacuoles and facilitates extensive bacterial replication in spacious vacuoles. Yet, the majority of infected host immune cells survive the infection and ultimately clear the bacteria. Thus, our data demonstrate that intra-vacuolar existence of Listeria monocytogenes in immune cells is not inevitably tied to bacterial persistence and chronic infection. We provide high resolution structural insights into how an effective inhibitory molecule interacts with the master regulator of Listeria virulence, which may be exploited for rational drug design.