Activation of the eis gene in a W-Beijing strain of Mycobacterium tuberculosis correlates with increased SigA levels and enhanced intracellular growth

286

311

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the ؊10 and ؊35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20 -180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 g/mL < minimum inhibitory concentration (MIC) <40 g/mL] but not to amikacin (MIC <4 g/mL). Additionally, 80% of clinical isolates examined in this study that exhibited low-level kanamycin resistance harbored eis promoter mutations. These results have important clinical implications in that clinical isolates determined to be resistant to kanamycin may not be cross-resistant to amikacin, as is often assumed. Molecular detection of eis mutations should distinguish strains resistant to kanamycin and those resistant to kanamycin and amikacin. This may help avoid excluding a potentially effective drug from a treatment regimen for drug-resistant TB.T he World Health Organization estimates that 9.2 million new cases of tuberculosis (TB) occur each year (1). Despite intensive efforts to ensure proper drug dosages and patient compliance with drug regimens, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis have emerged (2). These strains cause extensive mortality in immunocompromised individuals (3) and hinder the control and prevention of the disease. XDR and MDR TB infections cannot be adequately treated with the first-line anti-TB drugs and require expensive, prolonged treatment with second-line anti-TB drugs. The rapid determination of the resistance profile of an isolate can facilitate selection of an appropriate drug regimen and preclude development of additional drug resistances. Rapid detection of resistances is best achieved with molecular diagnostic approaches, particularly in developing countries where access to culture facilities is limited. Such strategies require a detailed understanding of the molecular basis for drug resistance. Although the mechanisms of resistance to first-line drugs such as isoniazid and rifampin are well characterized, much less is known about such mechanisms for the second-line drugs (4).An important second-line anti-TB drug is the aminoglycoside kanamycin (KAN), which binds to the 16S rRNA in the 30S ribosomal subunit and inhibits protein synthesis (5). In other bacteria, characterized mechanisms of KAN resistance include altered efflux or influx of the drug, inactivation of the drug by enzymatic modification, and mutation or methylation of rRNA, which disrupts binding of the drug to the riboso...

Section: Discussionmentioning

confidence: 55%

“…Recently, it was shown that sigA binds to the promoter region and regulates expression of the eis gene (15). Indeed, the Ϫ10 regions of the eis mutant promoters (C-14T, TACAGT; G-10A, CACAAT; C-12T, CAT-AGT) are more homologous with the SigA consensus sequence (TA(G/T)(A/G)AT) (16) than the WT promoter (CACAGT).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Zaunbrecher

Sikes²,

Metchock³

et al. 2009

286

311

“…The biological function of Eis in Mtb has been the subject of recent interest (6,7,(22)(23)(24)(25)(26)(27)(28)(29). The biochemical and structural studies described in this report provide clear evidence for an unprecedented multiacetylation capability of Eis that inactivates AG antibiotics.…”

mentioning

confidence: 75%

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

Chen

Biswas²,

Porter³

et al. 2011

106

260

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N -acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

“…The Mtb Eis protein is produced during human tuberculosis infection and is released into the culture medium (3). The sigma factor SigA was shown to bind to the eis promoter in the W-Beijing strain of Mtb, and the activation of the Mtb eis gene correlated with increased SigA levels and enhanced intracellular survival (13). Treatment of T cells with Mtb Eis inhibited ERK1/2, JAK pathway, and subsequent production of TNF-α and IL-4 (14).…”

mentioning

confidence: 99%

Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7

Kim¹,

Song³

et al. 2012

165

152

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N ɛ -acetyltransferase, rapidly acetylating Lys55 of dualspecificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N α -acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPSinduced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.Rv2416c | lysine acetylation | antituberculosis drug N early one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). This pathogenic bacterium causes tuberculosis, which claims the lives of millions of people every year (1). Tuberculosis has also become a global health issue owing to the increased incidences of multidrug-resistant and extensively drug-resistant strains of Mtb (2). This makes a search for targets of new antituberculosis drugs urgent. Mtb is a highly successful human pathogen, surviving and multiplying within the human macrophage cells of the infected people (3). Therefore, treatment of tuberculosis is difficult, requiring many months of taking a combination of antibiotics. Mtb has the ability to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis (4). Latent tuberculosis becomes activated when the body's immune system is weakened. As a result, tuberculosis is the major cause of death among immuno-compromised AIDS patients (5).In mycobacterial infection, host innate immune responses may play a crucial role in early protection against Mtb infection, leading to establishment of effective adaptive immunity to tuberculosis (6). Additionally, MAPK pathways are activated by Mtb or its components and play an e...