Ulrich Strych

Characterization of the Alanine Racemases from Pseudomonas aeruginosa PAO1

Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between l- and d-alanine. The requirement for d-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals. The dadXPA and alrPA genes have been sequenced, overexpressed, and their activity was demonstrated by complementing d-alanine auxotrophs of Escherichia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.

Status of vaccine research and development of vaccines for leishmaniasis.

A number of leishmaniasis vaccine candidates are at various stages of pre-clinical and clinical development. Leishmaniasis is a vector-borne neglected tropical disease (NTD) caused by a protozoan parasite of the genus Leishmania and transmitted to humans by the bite of a sand fly. Visceral leishmaniasis (VL, kala-azar) is a high mortality NTD found mostly in South Asia and East Africa, while cutaneous leishmaniasis (CL) is a disfiguring NTD highly endemic in the Middle East, Central Asia, North Africa, and the Americas. Estimates attribute 50,000 annual deaths and 3.3 million disability-adjusted life years to leishmaniasis. There are only a few approved drug treatments, no prophylactic drug and no vaccine. Ideally, an effective vaccine against leishmaniasis will elicit long-lasting immunity and protect broadly against VL and CL. Vaccines such as Leish-F1, F2 and F3, developed at IDRI and designed based on selected Leishmania antigen epitopes, have been in clinical trials. Other groups, including the Sabin Vaccine Institute in collaboration with the National Institutes of Health are investigating recombinant Leishmania antigens in combination with selected sand fly salivary gland antigens in order to augment host immunity. To date, both VL and CL vaccines have been shown to be cost-effective in economic modeling studies.

Mutant Analysis Shows that Alanine Racemases from Pseudomonas aeruginosa and Escherichia coli Are Dimeric

Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion.

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d-Alanine

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.

Neglected tropical diseases among the Association of Southeast Asian Nations (ASEAN): overview and update.

The ten member states of the Association of Southeast Asian Nations (ASEAN) constitute an economic powerhouse, yet these countries also harbor a mostly hidden burden of poverty and neglected tropical diseases (NTDs). Almost 200 million people live in extreme poverty in ASEAN countries, mostly in the low or lower middle-income countries of Indonesia, the Philippines, Myanmar, Viet Nam, and Cambodia, and many of them are affected by at least one NTD. However, NTDs are prevalent even among upper middle-income ASEAN countries such as Malaysia and Thailand, especially among the indigenous populations. The three major intestinal helminth infections are the most common NTDs; each helminthiasis is associated with approximately 100 million infections in the region. In addition, more than 10 million people suffer from either liver or intestinal fluke infections, as well as schistosomiasis and lymphatic filariasis (LF). Intestinal protozoan infections are widespread, while leishmaniasis has emerged in Thailand, and zoonotic malaria (Plasmodium knowlesi infection) causes severe morbidity in Malaysia. Melioidosis has emerged as an important bacterial NTD, as have selected rickettsial infections, and leptospirosis. Leprosy, yaws, and trachoma are still endemic in focal areas. Almost 70 million cases of dengue fever occur annually in ASEAN countries, such that this arboviral infection is now one of the most common and economically important NTDs in the region. A number of other arboviral and zoonotic viral infections have also emerged, including Japanese encephalitis; tick-borne viral infections; Nipah virus, a zoonosis present in fruit bats; and enterovirus 71 infection. There are urgent needs to expand surveillance activities in ASEAN countries, as well as to ensure mass drug administration is provided to populations at risk for intestinal helminth and fluke infections, LF, trachoma, and yaws. An ASEAN Network for Drugs, Diagnostics, Vaccines, and Traditional Medicines Innovation provides a policy framework for the development of new control and elimination tools. Together with prominent research institutions and universities, the World Health Organization (WHO), and its regional offices, these organizations could implement important public health improvements through NTD control and elimination in the coming decade.

Label-free, in situ SERS monitoring of individual DNA hybridization in microfluidics

We present label-free, in situ monitoring of individual DNA hybridization in microfluidics. By immobilizing molecular sentinel probes on nanoporous gold disks, we demonstrate sensitivity approaching the single-molecule limit via surface-enhanced Raman scattering which provides robust signals without photobleaching for more than an hour. We further demonstrate that a target concentration as low as 20 pM can be detected within 10 min under diffusion-limited transport.

New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

Background In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. Methodology/Principal Findings To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. Conclusions/Significance This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

New Vaccines for the World's Poorest People

The 2000 Millennium Development Goals helped stimulate the development of life-saving childhood vaccines for pneumococcal and rotavirus infections while greatly expanding coverage of existing vaccines. However, there remains an urgent need to develop new vaccines for HIV/AIDS, malaria, and tuberculosis, as well as for respiratory syncytial virus and those chronic and debilitating (mostly parasitic) infections known as neglected tropical diseases (NTDs). The NTDs represent the most common diseases of people living in extreme poverty and are the subject of this review. The development of NTD vaccines, including those for hookworm infection, schistosomiasis, leishmaniasis, and Chagas disease, is being led by nonprofit product development partnerships (PDPs) working in consortia of academic and industrial partners, including vaccine manufacturers in developing countries. NTD vaccines face unique challenges with respect to their product development and manufacture, as well as their preclinical and clinical testing. We emphasize global efforts to accelerate the development of NTD vaccines and some of the hurdles to ensuring their availability to the worlds poorest people.

Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13–50 nucleotide “guest” RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF “ribosomal RNA aptamer.” Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo. Copyright

Functionalized viral nanoparticles as ultrasensitive reporters in lateral-flow assays

Two types of viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). The particles were successfully integrated into an immunochromatographic assay detecting MS2 bacteriophage, a model for viral pathogens. The sensitivity of the assay was greatly superior to conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.