Targeting virulence: a paradigm for antimicrobial therapy [review] By Clathworthy, Pierson & Hung Alternative approach by targeting functions

s essential for infection, such as virulence factors required to cause host damage and disease In vivo viability experiment done in live isolated cells VS in vitro which takes place outside the living organism (i.e. test tube) Toxin function, toxin delivery, regulation of virulence expression, bacterial adhesion Inhibition of toxin function try to inhibit LF protease activity and promote cellular survival; interfere with toxin translocation by inhibiting formation of the pore by inhibiting endosome acidification; block downstream effects by targeting host proteins Targeting bacterial toxin delivery inhibit Type 3 and Type 2 secretion systems (needle-like apparatus) so that the virus will not be injected into the host organism Targeting regulation of virulence expression example of interfering with quorum sensing pathways, inhibiting bacterial populations ability to monitor the number of cells within that population; AHL-mediated quorum sensing disrupted > inhibit expression of genes; in vivo use of virstatin drug protected infant mice from intestinal colonization with V. cholerae Inhibition of adhesion proteins involved in bacterial adhesion are specific to prokaryotes; policies are aimed at inhibiting the formation of pili, makes it unable to grow pilin fiber, in vivo studies have shown to inhibit hemagglutination and biofilm formation in lab Virulence inhibitors as therapeutic candidates narrow spectrum and different mechanisms of action, inhibiting virulence would help preserve normal and potentially beneficial members of normal human microbiota; the time window for infection varies, so the mechanism for in vivo inhibition would be inhibitor-specific; also drugs would be more cost effective Future approaches to new antimicrobial development in vitro and in vivo bacterial essential gene functions are distinct; in vivo development most closely relates to the human environment because tests are conducted in living organisms Whole-organism based screening has the capacity to define which proteins are drug-targetable In vitro antimicrobial drug development is very inefficient, finding only 5 leads out of 70 cases Identification of new antimicrobials is likely to more effective in whole-cell based screens rather than in-vitro target based screens In vivo drugs could be developed relatively faster than in vitro drugs, which a range from 10-15 of development Tuberculosis: What We Dont Know Can, and Does, Hurt Us By Russell, Barry 3rd & Flynn Mycobacterium tuberculosis inadequate distribution of disease worldwide, increased drug resistance occurrence Life Cycle of M. tuberculosis infectious bacilli inhaled as droplet nuclei, invade the subtending epithelial layer which induces a localized inflammatory response. Granuloma is the building block of the pathogenic feature of tuberculosis; granulomas develop and eventually ruptures and spills thousands of viable, infectious bacilli into the airways Immune protection through vaccination progression to TB is linked to bacterial load; CD4 T cells are important in the control of human TB Bacillus Calmette-Guerin (BCG) only approved vaccine against TB: works in some populations but not in others 1. BCG too attenuated through culture 2. Exposure of infants to environmental mycobacteria (i.e. India) could lead to tolerance 3. Clearance of BCG may occur before development of a protective immune response Instead, you can overexpress mtb antigens, attenuating strains of mtb through gene deletion, and prime-boost strategies that direct and amplify an initial protective immune response Robust immune response is considered a major contributor to the pathology required for transmission of TB Variability between granulomas within a single infected person + highly differentiated internal structure = diverse range of environments for granuloma development Chemotherapeutic intervention requires over 6 months of treatment and current drugs used right now only target and kill the replicating organisms; the static, non replicating organisms would be latent and resistant; vaccinated mice showed that it had 1/10th the bacterial load when compared with the non-vaccinated, nave host Missing Tools the mouse model is different than the human immune response where granulomas are created; there is no noninvasive test for humans to detect the level of infection or progression to active disease Conclusions an effective infrastructure and healthcare system is required for TB prevention > failed treatment regimens, in combination with the absence of drug-resistance testing led to the selection and spread of lethal strains

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis (2009) By Zaunbrecher, Sikes, Metchock, Shinnick & Posey Abstract emergence of multidrug-resistant (MDR) tuberculosis (TB) is a growing worry, kanamycin and amikacin are two drugs used to treat MDR TB; however, they have identified mutations on the -10 and -35 promoter regions of the eis gene; the eis gene encodes for acetyltransferase; which in turn causes a 20-180 fold increase in eis leaderless mRNA transcript, which then leads to an increase in protein expression > 80% of the people studied had eis promoter mutation XDR is extensively drug resistant mycobacterium tuberculosis, along with MDR which is multi-drug resistant XDR + HDR = extensive mortality in immunocompromised individuals & hinder the control and prevention of TB Aminoglycoside kanamycin (KAN) = second line anti-TB drug [2nd to be prescribed by doctor] job is to bind to 16S chromosome in 30S ribosomal unit to inhibit protein synthesis Shown in other bacteria that KAN resistance by enzymatic modification or methylation of rRNA (disrupts binding of drug to the ribosome) Low-level resistance to KAN by causing overexpression of the enhanced intracellular survival (eis) protein, in M. tuberculosis C-14 mutation in eis (Rv2416c) confers KAN resistance and increases expression of eis to find out what strain is resistant to kanamycin, a cosmid library was formed using a plasmid that had the K204 gene sequence introduced to the H37Rv sequence which was then plated for KAN resistance; Reverse Transcriptase PCR assays showed that the sigA level was irrelevant when compared with mutant VS wild type strains; increase resistance to kanamycin when the C-14T gene was introduced and expressed; length of sequence (base pairs) also reinforces the increase of KAN resistance = eis transcripts also increased Transcription from the eis promoter produces a LEADERLESS mRNA by using RACE, rapid amplification of cDNA ends along with RT-PCR: this method allowed them to identify the start codon, GTG, and also that -10 and -35 regions of the eis promoter correspond with the location and mapping of the C-14T mutation Eis > proteins > aminoglycoside acetyltransferase > acetylates > inactivates KAN & AMK colorimetric assay that quantifies the conversion of acetyl-CoA into CoA-SH which leads to acetyltransferase activity (kanamycin resistance); if the gene lacked acetyl-CoA or the eis protein, the KAN would create a zone of inhibition, meaning that the antibodies were working against the gene, but when complete reactions were expressed, there was no ZOI = inactivation of both antibiotics = cell growth = KAN and AMK resistance There is a MIC, the minimum inhibitory concentration, which means that a minimum amount of drug is needed to inhibit growth of the cells Further DISCUSSION = FOCUS on the -10 and -35 regions of the promoter; suggests that both regions are required for optimal expression of the eis promoter; KAN and AMK resistance linked to observed low-level resistance > molecular level Immunoblot analysis used to see if the wild type or mutant with eis promoter could produce the eis protein under anti-eis protein conditions Leaderless mRNA lacked a 5' untranslated leader region that could contain a ribosomal binding site. They start directly at with the start codon.