Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 °C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cellbased system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4 ؉ T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.The ability of human immunodeficiency virus type 1 (HIV-1) to establish a latent infection results in life-long virus persistence even after long-term antiretroviral therapy (ART). 4 The role that latency plays in preventing sustained clearance of the virus infection has become evident in recent years. Patients that have been successfully treated with ART, having undetectable levels of viral RNA (below 50 copies/ml) in the plasma for years, experienced rapid virus rebound upon withdrawal of therapy (1, 2). Moreover, it was found that after T cell activation, virus could be isolated from CD4 ϩ T cells taken from these patients, underscoring the need to eliminate the latently infected cells to eradicate the virus (3-5).Activation of latent proviruses from infected cells in combination with ART is part of a therapeutic strategy that may lead to the complete elimination of HIV infection. Prior attempts to "flush out" the virus by activation of latently infected resting CD4 ϩ T cells with the administration of IL-2 and/or anti-CD3 monoclonal antibodies were ultimately unsuccessful, probably because of its inability to reach all of the latent viral reservoirs and the toxicity of the regimen (6 -10). A more promising approach to complete viral clearance is the use of small molecules with pharmacological properties that allow them to access the viral reservoirs and to specifically reactivate the latent proviruses. The concept of small molecule activation of latent HIV-1 has been tested in a clinical study using the histone deacetylase (HDAC) inhibitor valproic acid (VA) (11). However, it is questionable whether VA alone can be used as a supplement to ART for succe...

The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB–MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88DD-MyD88DD, MyD88DD-MyD88TIR and MyD88DD-MyD88 interactions but not MyD88–MyD88 or MyD88TIR-MyD88TIR interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD–TIR domain interface.

The rise in antimicrobial drug resistance, alongside the failure of conventional research to discover new antibiotics, will inevitably lead to a public health crisis that can drastically curtail our ability to combat infectious disease. Thus, there is a great global health need for development of antimicrobial countermeasures that target novel cell molecules or processes. RNA represents a largely unexploited category of potential targets for antimicrobial design. For decades, control of cellular behavior was thought to be the exclusive purview of protein-based regulators. The recent discovery of small RNAs (sRNAs) as a universal class of powerful RNA-based regulatory biomolecules has the potential to revolutionize our understanding of gene regulation in practically all biological functions. In general, sRNAs regulate gene expression by base-pairing with multiple downstream target mRNAs to prevent translation of mRNA into protein. In this review, we will discuss recent studies that document discovery of bacterial, viral, and human sRNAs and their molecular mechanisms in regulation of pathogen virulence and host immunity. Illuminating the functional roles of sRNAs in virulence and host immunity can provide the fundamental knowledge for development of next-generation antibiotics using sRNAs as novel targets.