Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid.

Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

Abstract Background A hallmark symptom of active pulmonary tuberculosis vital for disease transmission is cough. The current paradigm for tuberculosis-related cough is that it results from airway damage or irritation. However, there is limited experimental data to support this theory, and whether Mycobacterium tuberculosis (Mtb) induces cough to facilitate its own transmission has not been explored. The cough reflex is a complex and coordinated event involving both the nervous and musculoskeletal systems initiated by particulate or chemical molecules activating nociceptive neurons, which sense pain or irritation. This activation induces a signaling cascade ultimately resulting in a cough. Respiratory nociceptive neurons innervate the airway of humans and most mammals, and thus are poised to respond to noxious molecules to help protect the lung from damage. Because Mtb is a lung pathogen, cough is a primary mechanism of Mtb transmission, and respiratory nociceptive neurons activate cough, we hypothesized that Mtb produces molecules that stimulate cough, thereby facilitating its spread from infected to uninfected individuals. Methods We used an in vitro neuronal activation bioassay to fractionate, identify, and characterize Mtb cough-inducing molecules. We also measured cough in vivo in response to pure Mtb-derived cough molecules and during Mtb infection using a guinea pig model. Results We found that an acellular organic extract of Mtb triggers and activates nociceptive neurons in vitro with a neuronal response that is as robust as the response to capsaicin, an established nociceptive and cough-inducing molecule. Using analytical chemistry and our neuronal bioassay, we then isolated 2 molecules produced by Mtb that activate nociceptive neurons. Both the organic Mtb extract and purified molecules alone were sufficient to induce cough in a conscious guinea pig cough model. Finally guinea pigs infected with wild-type Mtb cough much more frequently than guinea pigs infected with Mtb strains unable to produce nociceptive molecules. Conclusion We conclude that Mtb produces molecules that activate nociceptive neurons and induce cough. These findings have significant implications for our understanding of Mtb transmission. Disclosures All authors: No reported disclosures.