David Alland, MD, MSc, DTM & H.
Padmini Salgame, PhD
Joel S. Freundlich, PhD
Jason H. Yang, PhD
Steven Sperber, MD
David Alland, MD, MSc, DTM & H.
The Research & Development Council of New Jersey hosted the 40th Annual Thomas Alva Edison Patent Awards at Liberty Science Center in Jersey City, New Jersey, on November 14, 2019. Rutgers, The State University of New Jersey and inventors David Alland and Soumitesh Chakravorty received a patent award in the Biotechnology category for “Detection of Drug Resistant Mycobacterium Tuberculosis”.
The Alland laboratory is interested in many different aspects of M. tuberculosis molecular biology, epidemiology and diagnostics. Many of these areas are related to understanding how antibiotic resistance develops on both the cellular and epidemiological level. We also have a major program in biodefense diagnostics and pathogenesis. Some specific areas of interest are described below.
Rapid diagnostics for biodefense. The advent of bioterrorism has placed a new emphasis on the need for rapid diagnostic assays that are sufficiently simple and robust that they can be performed in a doctor's office, clinic or local hospital. Blood cultures are one of the most common methods for detecting infections in patients with fever. A rapid "molecular" version of a blood culture would fulfill many of the diagnostic needs in health care settings for biodefense. The Alland laboratory has been working to develop a "molecular blood culture" that will make it possible to identify all common medical pathogens from blood samples in under an hour, without the need for conventional culture methods.
Intracellular consequences of antibiotic treatment. The anti-tuberculosis antibiotics isoniazid (INH) and ethambutol (EMB) fall into the general category of drugs that act by inhibiting components of cell wall biosynthesis. This class of antibiotics includes many of the most effective antibacterial agents. Significant progress has been made in identifying the targets that are inhibited by these antibiotics. Little is known, however, about the intracellular events that follow target inhibition. Expression profiling of INH-treated M. tuberculosis suggests that cell death is only the most apparent component of a series of intracellular events that occur following treatment with this antibiotic. This suggests that other cellular processes are induced, some of which may be involved in lysis and cell death (lytic factors), and others that may attempt to preserve cellular integrity and bacterial viability (protective factors). These events determine the ultimate fate of an antibiotic treated cell. The Alland laboratory has been working to understand the function of the pathways that are induced by antibiotic with a focus on the iniBAC genes ( Rv0341 , Rv0342 , Rv0343 ). We believe that a more full understanding of these pathways will lead to the discovery of important new antibiotics, including ones that act synergistically with currently available agents.
Studies of antibiotic resistance alleles in M. tuberculosis. Most causes of drug-resistance in M. tuberculosis appear to be the result of single nucleotide polymorphisms (SNPs) in particular target genes. However, each SNP occurs at relatively low frequency. Therefore, in the absence of large sequencing studies, it has been difficult to establish statistically valid associations between individual SNPs and resistance to a particular drug. In the case of resistance to the antibiotic isoniazid (INH), only mutations in codon 315 of the katG gene occur with sufficient frequency. The danger of reaching conclusions after analyzing only a limited number of M. tuberculosis isolates is illustrated by the case of SNPs in codon 463 of katG , and codons 269, and 312 of the kasA gene, which were originally identified as resistance associated, but were later shown to be common in INH-susceptible isolates. The Alland laboratory has developed several low-cost and high-thoughput methods to test clinical M. tuberculosis isolates for SNPs. We are currently in the process of analyzing over 2,000 samples for mutations that are possibly associated with antibiotic resistance. The goal of this project is to understand how different mutations arise, whether some mutations predispose or occur as the consequence of other mutations, and the effect of resistance mutations on further drug resistance evolution and disease transmission.
Evolutionary studies of M. tuberculosis. A microbial "species" often encompasses strains or related clones with distinct genotypic and phenotypic characteristics. Studies of epidemiology, pathogenesis, and immunity may depend on the ability to identify and subclassify these strains into related groups. Mycobacterium tuberculosis has been recalcitrant to clonal analysis because of its relatively low level of genetic polymorphism. IS6110 and other commonly used methods for M. tuberculosis strain typing are useful for epidemiological purposes. However, these methods are inadequate for testing hypotheses about the population and evolutionary genetics of the M. tuberculosis complex. Typing systems based on polymorphisms within variable-number tandem repeat regions address some but not all of these limitations. The lack of suitable genetic markers has made it nearly impossible to use specific subtypes of M. tuberculosis that exhibit defined biological and immunological characteristics in laboratory-based investigations. Clinical studies of pathogenesis, immunological responses, vaccines, and drug treatments have also been performed without accounting for the genotypic differences among the infecting M. tuberculosis strains. The deficiency of polymorphic genetic markers is critical because M. tuberculosis is not phenotypically and immunologically uniform. There is increasing evidence for variability in immunological and virulence characteristics. These differences include variations in resistance to effector immune molecules (such as reactive nitrogen intermediates), variations in resistance to killing by host cells, and differences in growth rate, lymphokine induction, and susceptibility to a vaccine primed host immune system. The Alland laboratory has pioneered a new strain typing system based on the use of synonymous SNPs as phylogenetic markers. We are currently using performing high-throughput analysis of M. tuberculosis populations from around the world to construct a SNP-based phylogenetic model of the M. tuberculosis species. This model will then assist us in investigating basic hypotheses about M. tuberculosis phylogenetics, evolution and epidemiology.
Integrated sample processing for detection of M. tuberculosis and the diagnosis of drug resistance. The rise of Multi-drug resistant (MDR) M. tuberculosis poses a special risk to populations with high prevalence of HIV infections. Nosocomial outbreaks of MDR tuberculosis are well documented in persons infected with HIV, and have occurred even in institutions with excellent infection control procedures. The risk of nosocomial spread of MDR tuberculosis is even higher in developing countries where patients with MDR tuberculosis may be cohorted with HIV positive patients. Simple, ultra-rapid methods to detect MDR tuberculosis are required to identify and segregate patients with MDR tuberculosis immediately upon admission to a health care setting. Conventional diagnostic and antibiotic drug susceptibility tests for M. tuberculosis infections are too insensitive and slow for this purpose, and even "rapid" methods cannot be performed in the short time required. This problem extends to PCR techniques because they are labor intensive and require technical sophistication, especially in the processing of samples and preparation of the assays. PCR applicability is also diminished by the presence of PCR inhibitors in clinical specimens and the risk of sample cross-contamination. We are working with Cepheid Inc. to develop a GeneXpert system to overcome all of these limitations. This system makes it possible to isolate and PCR amplify M. tuberculosis directly from sputum in a single hands-free step. Inexpensive molded-plastic cartridges concentrate all of the bacilli that are present in a sputum sample, remove PCR inhibitors, lyse the cells, and flush the resulting M. tuberculosis DNA into an integrated PCR reaction tube prefilled with dried real-time PCR reagents. Quantitative real-time PCR is performed automatically using a molecular beacon assay that detects approximately 95% of all mutations associated with rifampin resistance, a surrogate for MDR. The Alland laboratory has already completed the first stage of assay development. This system has been tested with a limited number of clinical sputum specimens, and it is performing well. We have been able to detect M. tuberculosis and MDR tuberculosis in less than one hour directly from sputum in a single hands-free step. We are now working to optimize the assay and then to begin clinical trials.
Roberto Colangeli, PhD
Hassan Safi, Ph.D.
Soumitesh Chakravorty, Ph.D.
Subramanya Srikar Lingaraju
Colangeli R, Haq A, Arcus V.L, Summers E, Magliozzo R. S, McBride A, Mitra A.K, Radjainia M, Khajo A, Jacobs Jr. W. R, Salgame P, Alland D. The multi-functional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates. Proc. Nat. Acad. Sci. U.S.A. 2009, 106:4414-4418. PMID: 19237572.
Tasslimi A, Sison E, Story E, Alland D , Burday M, Morrison S, Nalmas S, Smith S, Thomas PA, Wenger P, Sinha A. Disappearance of vaccine-type invasive pneumococcal disease (IPD) and emergence of serotype 19A in a minority population with high HIV prevalence and low childhood immunization rates. Clinical and Vaccine Immunology . 2009, 16 :1256-1259. PMID: 19515869
Abadia E, Sequera M, Ortega D, Méndez MV, Escalona A, Da Mata O, Izarra E, Rojas Y, Jaspe R, Motiwala AS, Alland D , de Waard JH, Takiff HE. Mycobacterium tuberculosis ecology in Venezuela : Epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-24. BMC Infectious Diseases. 2009, 9 :122. PMID: 19660112.
Safi H, Fleischmann RD, Peterson SN, Jones MB, Jarrahi B, Alland D. Allelic exchange and mutant selection demonstrate that common clinical embCAB gene mutations only modestly increase resistance to ethambutol in Mycobacterium tuberculosis. Antimicrob Agents Chemother . 2010, 54 :103-108. PMID: 19822701.
Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, Kop J, Owens MR, Rodgers
R, Banada P, Safi H, Blakemore R, Lan NT, Jones-López EC, Levi
M, Burday M, Ayakaka I, Mugerwa RD, McMillan B, Winn-Deen E, Christel
L, Dailey P, Perkins MD, Persing DH, Alland D . Rapid
detection of Mycobacterium tuberculosis and rifampin-resistance using
on-demand, near patient technology. J Clin Microbiol . 2010,
48 :229-237. PMID: 19864480.
Chakravorty S, Aladegbami B, Burday M, Levi M, Marras SA, Shah D, El-Hajjj HH, Kramer
FR, Alland D . Rapid Universal Identification of bacterial
pathogens from clinical cultures by using a novel sloppy molecular beacon
melting temperature signature technique. J Clin. MIcrobiol .
2010, 48 :258-267 PMID 19923485
Motiwala AS, Dai Y, Jones EC, Hwang SH, Lee JS, Cho SN, Via LE, Barry CE 3 rd
, Alland D . Mutations in extensively drug-resistant
Mycobacterium tuberculosis that do not code for known drug-resistance
mechanisms. J Infect Dis. 2010, 201:881-8. PMID: 20136412
Blakemore R, Story E, Helb D, Kop J, Banada P, Owens MR, Chakravorty S, Jones
M, Alland D . Evaluation of the analytical performance
of the Xpert MTB/RIF assay. J Clin Microbiol . 2010 48
:2495-501. PMID: 20504986.
Wang F, Jain P, Gulten G, Liu Z, Feng Y, Ganesula K, Motiwala AS, Ioerger
TR, Alland D , Vilchèze C, Jacobs WR Jr, Sacchettini
JC. Mycobacterium tuberculosis Dihydrofolate reductase is
not a target relevant to the anti-tubercular activity of isoniazid.
Antimicrob agents chemother. 2010 Jun 21. [Epub ahead of print]
|Low-Cost Integrated Molecular Assay System for the Detection of TB Drug Resistance
|Integrated Dual-Use Systems for Bio-Defense & Sepsis Diagnosis (ARRA)-Equipment
|Rapid Diagnosis of XDR TB
|Development of a Second Generation MDR-XDR TB Assay
|Xpert TB Diagnostic Project
||Foundation for Innovative New Diagnostics (FIND)
|Integrated Dual-Use Systems for Bio-Defense & Sepsis Diagnosis (ARRA)
|Mycobacterium TB Biomarkers for Diagnosis + Care
||Catalysis Foundation for Health
|TB Clinical Diagnostics Research Consortium
||John Hopkins University
|Memory Immunity in TB
||American Heart Association.
Host IMMUNE Responses in Tuberculosis
of the research programs of my laboratory is focused on elucidating
the mechanistic principles that govern Th1 initiation in tuberculosis.
We are studying the interplay between dendritic cells, macrophages,
and T cells through the use of transgenic model systems to define the
role of these cells in initiation of Th1 immunity. Previously we had
demonstrated that dendritic cells produce Interleukin (IL)-12, a Th1
driving cytokine, in response to M. tuberculosis stimulii
alone. In contrast, IL-12 induction from macrophages required T cell-derived
signals, in addition to microbial trigger. Studies examining the molecular
basis for differential IL-12 regulation in M. tuberculosis
stimulated dendritic cells and macrophages are in progress. We are also
studying the reprogramming of the dendritic cell and macrophage transcriptome
in response to M. tuberculosis infection to better understand
the role of the two cell types in initiating Th1 immunity in tuberculosis.
My laboratory is also examining the role of these two antigen-presenting
cell types in orchestrating the formation of granuloma during tuberculous
resulting from CD95 is a feature of T cells that have been repeatedly
stimulated through their antigen-specific receptor, and may be a fail-safe
mechanism to prevent excess T cell activation. Previous work from our
laboratory has established that Th1 cells exhibit pronounced susceptibility
to activation-induced CD95-mediated apoptosis, and in contrast Th2 cells
are resistant. We have demonstrated that mechanistically the difference
in sensitivity to apoptosis of the two subsets is due to a selective
lack of upregulation of phosphatidylinositol 3'-kinase activity in Th1
cells following activation. Studies to determine the molecular mechanisms
for the differential response by Th1 and Th2 cells to receptor activation,
and its effect on CD95 death pathway are ongoing. Additionally, we are
also investigating whether M. tuberculosis establishes persistence
in the host by interfering in the cross talk between CD3/TCR and CD95
signaling pathways of Th1 cells. Specifically, we are testing the hypotheses
that in tuberculous infection, CD95-mediated Th1 depletion occurs, resulting
in attenuation of protective immunity against M. tuberculosis.
Furthermore, we are also actively investigating in patients with pleural
and lymphadenitis tuberculosis whether Th1 apoptosis contributes to
we hope that paradigms developed from our studies will provide innovative
strategies for vaccine development and also provide new modalities of
modulating the immune response to shorten chemotherapy and/or overcome
Kamlesh Bhatt, Ph.D.
Wasiulla Rafi, Ph.D.
Aleksandra Uzelac, B.S.
Chenggang Hu, C., Mayadas-Norton, T., Chan, J., Tanaka, K., Salgame, P. Mycobacterium tuberculosis infection in complement receptor 3 deficient mice. J. Immunol. 165:2596, 2000.
Varadhachary, A.S., Edidin, M., Peter, M.E., Krammer, P.H., Salgame, P. Phosphatidylinositol 3'-kinase protects Th2 cells from apoptosis by modulating lateral diffusion and aggregation of CD95. J. Immunol. 166:6564-6569, 2001.
Hickman, S., Chan, J., Salgame, P. Mycobacterium tuberculosis
induces differential cytokine production from dendritic cells and macrophages
with divergent effects on naive T cell polarization. J. Immunol. 168:4636-4642,
K., Perdow Hickman, S. and Salgame, P. A new approach to modeling early
lung immunity in murine tuberculosis. Cutting Edge: J. Immunol. 172:2748-2751,
S., Uematsu, S., Akira, S. and Salgame, P. IL-6 and IL-10 induction
from dendritic cells in response to Mycobacterium tuberculosis
is predominantly dependent on Toll-like receptor 2-mediated recognition.
J. Immunol. 173:3392-3397, 2004.
P. Host innate and Th1 responses and the bacterial factors that control
Mycobacterium tuberculosis infection. Current Opinion in Immunol.
L., Jang, S., Zamlynny, B., Ravikumar, S., McBride, A., Hickman , S.
P. and Salgame, P. Disparity in Interleukin-12 release in dendritic
cells and macrophages in response to Mycobacterium tuberculosis
is due to utilization of distinct Toll-like receptors. J. Immunol.
K. and Salgame, P. Innate Immune Response in Mycobacterial infections.
The Journal of Clinical Immunology. 27:347-62, 2007.
S., Uzelac, A. and Salgame, P. Distinct chemokine and cytokine gene
expression pattern of murine dendritic cells and macrophages in response
to Mycobacterium tuberculosis infection. J. Leucocyte Biol.
84: 1264-1270, 2008.
K., Uzelac, A., Mathur, S., McBride, A., Potian, J. and Salgame , P.
B7 costimulation is critical for host control of chronic M. tuberculosis
infection. J. Immunology, 182:3793-3800, 2009.
|Tubercle Granuloma & Memory Immunity
|Systems Biology Approach to the Mechanisms of TB Latency + Reactivation
|TLR2 and the Tubercle Granuloma
| Heminth Modulation of Mtb
Joel S. Freundlich, PhD
Joel S. Freundlich, Ph.D., is an Associate Professor of Pharmacology, Physiology & Neuroscience and of Medicine at Rutgers University–New Jersey Medical School. He arrived at NJMS in 2011, and is a member of the Division of Infectious Diseases and The Center for Emerging and Re-emerging Pathogens. Prior to his return to academic research, he spent eight years in the pharmaceutical industry as a medicinal chemist. His undergraduate and master’s degree training were in chemical engineering at Cornell University as a McMullen Dean’s Scholar. He received his doctorate in organic chemistry from the Massachusetts Institute of Technology under the tutelage of 2005 Nobel Prize in Chemistry awardee Richard Schrock.
See his lab website at: http://njms.rutgers.edu/departments/labs/freundlich/index.cfm
His laboratory focuses on the following:
A. Novel chemical probes of M. tuberculosis discovered through innovative methods
We assert that the research community can more optimally leverage the large data sets created over the last ca. 70 years of exploring small molecule inhibition of the growth and/or killing of M. tuberculosis. In vitro and in vivo data may be utilized to create machine-learning models. These models provide highly valuable characterizations of the physiochemical properties and structural features of both active and inactive molecules. They also enable the successful prediction (hit rates often ~ 10 – 20%) of novel antituberculars that have significantly different structures than the training set used to educate the model. Thus, they enable exploration of new chemical space more efficiently. We believe that such molecules, given their novel chemotypes amongst antituberculars, have a heightened probability of modulating novel biological targets (See B). Thus, novel computational methods are being developed and then implemented to uncover new chemical probes and drug discovery hits/leads.
These novel antituberculars are then optimized with respect to their efficacy, in vitro Absorption-Distribution-Metabolism-Excretion, in vivo pharmacokinetic, and toxicity profiles to obtain high-value chemical probes and drug discovery hits/leads.
Representative Papers: Pharm Res 2016, PLoS One 2015, J Chem Inf Model 2014, Pharm Res 2014, J Chem Inf Model 2013, PLoS One 2013, Chem Biol 2013, Pharm Res 2012, Pharm Res 2011, mBio 2011.
B. Exploration of the polypharmacology of novel chemical probes and what it reveals about new strategies to pursue synergistic targets
Given our unique mixture of experience with both academic tuberculosis research and industrial drug discovery, we are particularly fascinated by polypharmacology. Polypharmacology may be defined as the ability of a single small molecule to modulate multiple drug targets within a pathogen to elicit a favorable therapeutic effect. We assert that this is not rampant promiscuity, but it can instead represent the beneficial perturbation of targets within the same pathway or disparate pathways. Utilizing a range of biological techniques, including transcriptomics, metabolomics, and drug resistance studies, we aim to elucidate how a select set of novel antituberculars utilize polypharmacology to achieve significant cidal activity versus M. tuberculosis. These multidisciplinary studies can lead to the identification of novel combinations of targets that can be pursued to develop new multi-target inhibitors or new synergistic combination therapies.
Representative Papers: Tet Lett 2015, Chem Biol 2014, Antimicrob Agents Chemother 2011, Tuberculosis 2010.
C. Extension of our computational and chemical discovery platforms to other infectious diseases
The Freundlich lab also seeks to extend and apply our computational and chemical techniques to enhance the efficiency of drug discovery research against other infectious diseases. We view these methodologies as being critical to furthering our basic understanding of the biology of bacteria and viruses of global health relevance, while also seeding drug discovery efforts. Through our NIH/NIAID-funded U19 program, we are working on the ESKAPE bacteria. In 2016, we launched a new collaboration with IBM, Dr. Carolina Horta Andrade, and Dr. Sean Ekins that is focused on jump-starting drug discovery efforts against the Zika virus, by performing massive virtual screens on the World Community Grid. We have also published on computational approaches, which have suggested potential starting points for an Ebola virus therapy.
Representative Papers: F1000Research 2016, F1000Research 2015, PLoS One 2014, Pharm Res 2014.
For details on current members of the Freundlich lab and the alumni, see:
Jason H. Yang, PhD
The Yang Lab at Rutgers New Jersey Medical School seeks to understand the molecular mechanisms underlying the pathogenesis and treatment of chronic and infectious diseases. We develop approaches for accelerating our causal understanding of biological systems, integrating network modeling with machine learning and high-throughput experimentation.
Our overall goal is to tackle the most important challenges in global health with systems approaches that can enable new and innovative therapeutics.
The Yang Lab
Steven Sperber, MD
Dr. Sperber's major research interests include general infectious diseases, respiratory viruses and infections due to multi-drug resistant bacteria. Dr. Sperber has recently collaborated on several multi-center projects to better define the scope of infections caused by multi-drug resistant gram negative rods.
Satlin MJ, Chen L, Patel G, Gomez-Simmonds A, Weston G, Kim AC, Seo SK, Rosenthal ME, Sperber SJ, Jenkins SG, Hamula CL, Uhlemann A-C, Levi, MH, Fries BC, Tang Y-W, Juretschko S, Rojtman AD, Hong T, Mathema B, and Jacobs MR. Bacteremia due to carbapenem-resistant Enterobacteriaceae (CRE): A multicenter clinical and molecular epidemiologic analysis in the nation’s epicenter for CRE. Antimicrob Agents Chemother 2017; 61:e02349-16.
Imran TF, Yick F, Dhamotharan S, and Sperber SJ. Emphysematous pyelonephritis with abscess masquerading as an ordinary urinary tract infection. Am J Infect Dis 2015;11:88-92.
Satlin M, …Sperber S…. Impact of Rapid Diagnostics and Ceftazidime–Avibactam on Mortality after Bacteremia Caused by Carbapenem-Resistant Enterobacteriaceae. Presented at ID Week 2019, Washington, D.C , October 4, 2019 OFID 2019: 6 (Suppl 2), S41.