Christine L. Cosma

Drug Tolerance in Replicating Mycobacteria Mediated by a Macrophage-Induced Efflux Mechanism

Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multidrug-tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug or more specific efflux pump inhibitors to standard antitubercular therapy should shorten the duration of curative treatment.

Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.

The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.

Superinfecting mycobacteria home to established tuberculous granulomas

A central paradox of tuberculosis immunity is that reinfection and bacterial persistence occur despite vigorous host immune responses concentrated in granulomas, which are organized structures that form in response to infection. Prevailing models attribute reinfection and persistence to bacterial avoidance of host immunity via establishment of infection outside primary granulomas. Alternatively, persistence is attributed to a gradual bacterial adaptation to evolving host immune responses. We show here that superinfecting Mycobacterium marinum traffic rapidly into preexisting granulomas, including their caseous (necrotic) centers, through specific mycobacterium-directed and host cell–mediated processes, yet adapt quickly to persist long term therein. These findings demonstrate a failure of established granulomas, concentrated foci of activated macrophages and antigen-specific immune effector cells, to eradicate newly deposited mycobacteria not previously exposed to host responses.

Zebrafish and Frog Models of Mycobacterium marinum Infection

Mycobacterium marinum infection of poikilothermic animals, such as fish and frogs, results in chronic granulomatous diseases that bear many similarities to mycobacterioses in mammals, including tuberculosis. This unit describes three animal models of M. marinum infection that can be used to study basic aspects of Mycobacterium‐host interactions and granuloma development, as well as trafficking of immune cells in host tissues. Protocols are included that describe intraperitoneal infection of adult leopard frogs (Rana pipiens) and zebrafish (Danio rerio). Protocols also describe subsequent monitoring of the infection by enumeration of bacterial cfu, mean time to death, or visual examination of infected tissue using both conventional histological stains and fluorescence microscopy of fluorescently marked bacteria. Furthermore, protocols are included that describe the infection of embryonic zebrafish and the subsequent analysis of the infection in real time using DIC and fluorescence microscopy.

Trafficking of Superinfecting Mycobacterium Organisms into Established Granulomas Occurs in Mammals and Is Independent of the Erp and ESX-1 Mycobacterial Virulence Loci

Although tuberculous granulomas, which are composed of infected macrophages and other immune cells, have long been considered impermeable structures, recent studies have shown that superinfecting Mycobacterium marinum traffic rapidly to established fish and frog granulomas by host-mediated and Mycobacterium-directed mechanisms. The present study shows that superinfecting Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin similarly home to established granulomas in mice. Furthermore, 2 prominent mycobacterial virulence determinants, Erp and ESX-1, do not affect this cellular trafficking. These findings suggest that homing of infected macrophages to sites of infection is a general feature of the pathogenesis of tuberculosis and has important consequences for therapeutic strategies.

An In Vivo Platform for Rapid High-Throughput Antitubercular Drug Discovery

Treatment of tuberculosis, like other infectious diseases, is increasingly hindered by the emergence of drug resistance. Drug discovery efforts would be facilitated by facile screening tools that incorporate the complexities of human disease. Mycobacterium marinum-infected zebrafish larvae recapitulate key aspects of tuberculosis pathogenesis and drug treatment. Here, we develop a model for rapid in vivo drug screening using fluorescence-based methods for serial quantitative assessment of drug efficacy and toxicity. We provide proof-of-concept that both traditional bacterial-targeting antitubercular drugs and newly identified host-targeting drugs would be discovered through the use of this model. We demonstrate the models utility for the identification of synergistic combinations of antibacterial drugs and demonstrate synergy between bacterial- and host-targeting compounds. Thus, the platform can be used to identify new antibacterial agents and entirely new classes of drugs that thwart infection by targeting host pathways. The methods developed here should be widely applicable to small-molecule screens for other infectious and noninfectious diseases.

Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo

Summary The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection.

10 for 10

Cell Host & Microbe is 10! In March, we marked the anniversary with storytelling—looking back at some of the seminal papers published in the journal and how they came to be. Now, for this issue, as a continuation of the 10th anniversary festivities, we invited 10 Cell Host & Microbe authors, who are experts in their field, to join us in the celebrations by lending their “Voice”—each reflecting on an area of host-microbe research that the journal has contributed to advancing. These Voices (pp. 130–133) echo the spirit of the journal; they highlight the breadth of science the journal has covered in these 10 years and some of the scientific areas we have sought to impact. They also reflect on our ethos—fostering the intermingling of fields, embracing new fields and ideas, banishing dogmas, pushing boundaries, and seeking the leading edge.We also present a collection of 10 Reviews and Perspectives that highlight broad themes in host-microbe biology from stalwarts in their fields. A pair of Perspectives by Jeffrey Gordon and colleagues (pp. 134–141) as well as Julia Vorholt et al. (pp. 142–155) examine how microbiome communities can be examined and harnessed to maximize human nutrient absorption and plant health. The role of commensal fungi in health and disease is then reviewed by David Underhill and colleagues (pp. 156–165). David Holden and colleagues (pp. 217–231) examine recent insights into how Salmonella effectors contribute to the virulence and survival of this intracellular pathogen, while Gabriel Mitchell and Ralph Isberg (pp. 166–175) describe the interplay between host innate immune responses that balances pathogen elimination with inflammation. Another host defense strategy mediated by the interferon response is discussed by Adolfo Garcia-Sastre (pp. 176–184), along with virus strategies to counteract these protective mechanisms. As reviewed by Eran Elinav, Paolo Sassone-Corsi, and colleagues (pp. 185–192), many of these antimicrobial responses are regulated by the circadian clock to maximize the host defense against pathogenic attack. The timing and sequence of molecular events in Plasmodium invasion of erythrocytes is also critical to this host-microbe exchange, as discussed by Alan Cowman et al. (pp. 232–245). Finally, antiviral neutralizing antibodies take the stage with reviews by Yoshiaki Nishimura and Malcolm Martin (pp. 207–216) examining their immunotherapeutic efficacy in HIV and James Crowe (pp. 193–206) examining how neutralizing antibodies can be studied and generated to harness their power for vaccines.This is also a good time to reflect on the journal’s growth—a chance to stand the “child” against the growth chart and see how she has grown. And how she has grown! When the journal was initially announced, we heard from a few naysayers: Do we really need another journal? Don’t we have enough microbiology journals? In explaining the journal’s mission of finding common ground in the interactions of different microbes with their host, the counter-argument we heard was that each pathogen has evolved unique mechanisms to interact with the host, and therefore there would be no common ground. However, we believed that because the microbe has to deal with defined host mechanisms and pathways (e.g., the epithelial barrier, innate immunity, etc.), we could learn a lot about the host from each microbe, and vice versa. In hindsight, we now see that common ground is the rule rather than the exception, and that evolution has repeatedly employed common strategies in allowing microbes to manipulate their host systems, and conversely, for host organisms to control against and select for the microbes they encounter. These commonalities have been made clear by researchers who think broadly in the interpretation of their findings in the context of other systems, and it is these kinds of studies that we have been privileged to publish. We also hope that in some way we have been able to promote this broad thinking by bringing together a community of authors and readers to the journal.We thank our authors in this issue for contributing thought-provoking and timely pieces and all of our authors, reviewers, and readers over the past 10 years for their interest, contributions, and support. We hope that you will enjoy our “10 for 10” features, and we look forward with great excitement to serving the community for the next 10 years. Please send us your feedback by engaging via Twitter (@cellhostmicrobe) or by email (hostmicrobe@cell.com).