The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis (TB), 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 tissues1. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show 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) lipids2 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 (PGL)2, promote recruitment of permissive macrophages via a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence3 and PGL, which (along with CCR2) is known to be associated with human TB4,5. 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 signaling.

DegP is a heat-shock inducible periplasmic protease in Escherichia coli. Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by or32.Rather, the degP gene is transcriptionally regulated by an alternate heat shock or factor, orE. Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter. To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion. Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP. Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP. These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein. Cpx-mediated transcription of degP is partially dependent on the activity of Eor E, suggesting that the Cpx pathway functions in concert with Eor E and perhaps other RNA polymerases to drive transcription of degP.[Key Words: Heat shock; (rE; receptor kinase; lipoprotein; response regulator] Received November 23, 1994; revised version accepted January 12, 1995.The heat shock, or stress, proteins are a ubiquitous set of proteins whose synthesis is induced in response to environmental insults such as abrupt temperature elevation. It is thought that during times of stress these proteins maintain viability of the cell by degrading proteins that have been irreversibly inactivated and by promoting the renaturation/activation of reversibly inactivated proteins. Thus, heat shock proteins are often proteases or molecular chaperones (Gething and Sambrook 1992;Bukau 1993;Craig et al. 1993). Although heat shock proteins are required during times of stress, many of them also perform important functions in unstressed cells. For example, in Escherichia coli the molecular chaperones are thought to assist in protein folding (Zeilstra-Ryalls et al. 1991;Gething and Sambrook 1992), whereas proteases such as Lon serve post-translational regulatory roles (Gottesman 1989;Goldberg 1992}. Because heat shock proteins perform such fundamental functions, it is not surprising that they are found in a variety of subcellular compartments in both prokaryotic and eukaryotic cells (Deshaies et al. 1988;Strauch and Beckwith 1988; Craig et al. 1989;Rose et al. 1989). Interestingly, the regulation of stress proteins found in one compartment is often coordinated independently of stress proteins within other compartments (Strauch and Beckwith 1988;Strauch et al. 1989;Mori et al. 1993). For example, stresses that specifically perturb the bacterial envelope in E. coli increase the synthesis of the periplasmic protease DegP (Lipinska et al. 1990), whereas the synthesis of cytoplasmic stress proteins remains unaffected (Mecsas et a...

SUMMARY 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 multi-drug 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 newly discovered 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 inhibitors, to standard antitubercular therapy may shorten the duration of curative treatment.

Pathogenic mycobacteria, including the causative agents of tuberculosis and leprosy, are responsible for considerable morbidity and mortality worldwide. A hallmark of these pathogens is their tendency to establish chronic infections that produce similar pathologies in a variety of hosts. During infection, mycobacteria reside in macrophages and induce the formation of granulomas, organized immune complexes of differentiated macrophages, lymphocytes, and other cells. This review summarizes our understanding of Mycobacterium-host cell interactions, the bacterial-granuloma interface, and mechanisms of bacterial virulence and persistence. In addition, we highlight current controversies and unanswered questions in these areas.

Identification of host factors that interact with pathogens is crucial to an understanding of infectious disease, but direct screening for host mutations to aid in this task is not feasible in mammals. The nematode Caenorhabditis elegans is a genetically tractable alternative for investigating the pathogenic bacterium Pseudomonas aeruginosa. A P. aeruginosa toxin, produced at high cell density under control of the quorum-sensing regulators LasR and RhlR, rapidly and lethally paralyzes C. elegans. Loss-of-function mutations in C. elegans egl-9, a gene required for normal egg laying, confer strong resistance to the paralysis. Thus, activation of EGL-9 or of a pathway that includes it may lead to the paralysis. The molecular identity of egl-9 was determined by transformation rescue and DNA sequencing. A mammalian homologue of EGL-9 is expressed in tissues in which exposure to P. aeruginosa could have clinical effects. T he use of genetic strategies to identify host factors with which pathogens interact has been limited by the fact that traditional disease model hosts, live mammals or their cultured cells, are not amenable to facile genetic analysis. Invertebrate models, such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, offer sophisticated genetic methods but have rarely been used to study infectious disease. Complete sequencing of the genome of C. elegans indicated that at least 36% of the 19,000 predicted proteins have matches in humans (1). Thus, despite the enormous evolutionary distance between humans and nematodes, C. elegans may be a valid model for studies of numerous disease processes.Pseudomonas aeruginosa is an opportunistic pathogen that infects patients compromised by illness, injury, or inborn genetic defect (2, 3). Hospital-acquired P. aeruginosa pneumonias and septicemias are frequently lethal, and the bacteria infect the lungs of the majority of patients with cystic fibrosis. P. aeruginosa secretes numerous protein virulence factors, including ADP ribosylating enzymes, proteases, and phospholipases, as well as small molecules that include phenazines, rhamnolipid, and cyanide. Production of some of these factors is controlled by one or both of two quorum-sensing systems, LasI͞R and RhlI͞R, which tie gene expression to high cell density (4).Recently, Ausubel and colleagues (5-7) described a model for P. aeruginosa pathogenesis that uses C. elegans. Depending on the experimental conditions, strain PA14 kills the nematodes over a period of days (slow killing) or hours (fast killing). The fast killing is mediated by the production of phenazine compounds, which may act through the generation of active oxygen species.Using a different strain of P. aeruginosa (8), we have identified a third mode by which this species can kill C. elegans. The lethal effect is associated with a rapid neuromuscular paralysis and is caused by the action of one or more diffusible factors whose production requires the Las and Rhl quorum-sensing systems. C. elegans strains carrying loss-of-func...