Publications by authors named "Thomas A Ficht"

53 Publications

Interactions between fungal hyaluronic acid and host CD44 promote internalization by recruiting host autophagy proteins to forming phagosomes.

iScience 2021 Mar 12;24(3):102192. Epub 2021 Feb 12.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX 77807, USA.

Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.
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http://dx.doi.org/10.1016/j.isci.2021.102192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920835PMC
March 2021

Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats.

Vaccine 2021 01 13;39(3):617-625. Epub 2020 Dec 13.

Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA. Electronic address:

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x10 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.
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http://dx.doi.org/10.1016/j.vaccine.2020.11.033DOI Listing
January 2021

The candidate vaccine strain Brucella ovis ∆abcBA is protective against Brucella melitensis infection in mice.

Microbiol Immunol 2020 Nov 8;64(11):730-736. Epub 2020 Oct 8.

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.
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http://dx.doi.org/10.1111/1348-0421.12850DOI Listing
November 2020

Protective antibody response following oral vaccination with microencapsulated Sterne strain 34F2 spores.

NPJ Vaccines 2020 10;5:59. Epub 2020 Jul 10.

Texas A&M University, Department of Veterinary Pathobiology, College Station, TX 77843 USA.

An oral vaccine against anthrax () is urgently needed to prevent annual anthrax outbreaks that are causing catastrophic losses in free-ranging livestock and wildlife worldwide. The Sterne vaccine, the current injectable livestock vaccine, is a suspension of live attenuated Sterne strain 34F2 spores (Sterne spores) in saponin. It is not effective when administered orally and individual subcutaneous injections are not a practical method of vaccination for wildlife. In this study, we report the development of a microencapsulated oral vaccine against anthrax. Evaluating Sterne spore stability at varying pH's in vitro revealed that spore exposure to pH 2 results in spore death, confirming that protection from the gastric environment is of main concern when producing an oral vaccine. Therefore, Sterne spores were encapsulated in alginate and coated with a protein shell containing poly-L-lysine (PLL) and vitelline protein B (VpB), a non-immunogenic, proteolysis resistant protein isolated from . Capsule exposure to pH 2 demonstrated enhanced acid gel character suggesting that alginate microcapsules provided the necessary protection for spores to survive the gastric environment. Post vaccination IgG levels in BALBc/J mouse serum samples indicated that encapsulated spores induced anti-anthrax specific responses in both the subcutaneous and the oral vaccination groups. Furthermore, the antibody responses from both vaccination routes were protective against anthrax lethal toxin in vitro, suggesting that further optimization of this vaccine formulation may result in a reliable oral vaccine that will conveniently and effectively prevent anthrax in wildlife populations.
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http://dx.doi.org/10.1038/s41541-020-0208-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351773PMC
July 2020

A Tractable Cell System Enables Rapid Identification of Host Factors.

Front Cell Infect Microbiol 2020 26;10:240. Epub 2020 May 26.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan TX, United States.

is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported. is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.
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http://dx.doi.org/10.3389/fcimb.2020.00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264411PMC
June 2021

Vaccine Candidate Brucella melitensis 16M Is Safe in a Pregnant Sheep Model and Confers Protection.

mSphere 2020 05 13;5(3). Epub 2020 May 13.

College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA

As a natural host species for , pregnant sheep offer an ideal model to evaluate vaccine candidates for safety. strain Rev. 1 has been used almost exclusively to prevent brucellosis in small ruminants, but it causes abortions when given to pregnant animals. To evaluate the comparative safety of the candidate 16M, pregnant sheep ( = 6) were vaccinated subcutaneously with 1 × 10 CFU/ml of 16M or 1 × 10 CFU/ml Rev. 1 at a highly susceptible stage of gestation (approximately 70 days). 16M resulted in only 1 abortion (1 of 6) compared with 4 of 6 (66.7%) abortions in the Rev. 1 cohort. The placenta was evaluated by culture to determine if vaccination resulted in colonization. As another measure of safety, effects of on the fetus/offspring (vertical transmission) was evaluated by culture and histopathology of fetal tissues to determine if vaccination prevented infection of the fetus. Vaccination with 16M resulted in less vertical transmission than Rev. 1. To determine if vaccination was efficacious and could reduce tissue colonization in sheep, the same cohort of sheep were challenged 5 weeks postpartum by conjunctival inoculation with 1 × 10 CFU/ml Protection was similar between Rev. 1 and 16M, with no statistical difference in colonization in the target organs. Overall, the 16M vaccine was considered safer than Rev. 1 based on a reduced number of abortions and limited infection in the offspring. Future experiments are needed to further refine the vaccine dose to increase the safety margin and to evaluate protection in pregnant ewes. Brucellosis is one of the most commonly reported zoonotic disease with a worldwide distribution. Of the 12 species, is considered the most virulent and causes reproductive failure (abortions/stillbirths) in small ruminants, which can spread the disease to other animals or to humans. Vaccination of small ruminants is a key measure used to protect both human and animal health. However, the commercially available live-attenuated vaccine for Rev. 1 retains virulence and can cause disease in animals and humans. In order to evaluate the safety and efficacy in sheep, we vaccinated pregnant sheep with 16M Our results indicate that 16M was safer for use during pregnancy, provided a similar level of protection as Rev. 1, and could be considered an improved candidate for future vaccine trials.
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http://dx.doi.org/10.1128/mSphere.00120-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227765PMC
May 2020

Vaccine safety studies of S19 and S19Δ in pregnant swine.

Vaccine X 2019 Dec 22;3:100041. Epub 2019 Aug 22.

Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical, Sciences, Texas A&M University, College Station, TX, USA.

Brucellosis in swine is caused by a bacterial infection of nearly worldwide distribution. is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19Δ. As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ± 0.508 × 10 CFU of either S19 or S19Δ. Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti- IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4-6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine.
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http://dx.doi.org/10.1016/j.jvacx.2019.100041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737346PMC
December 2019

The NOD- Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety.

Infect Immun 2019 06 21;87(6). Epub 2019 May 21.

Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA

Osteoarticular brucellosis is the most common complication in -infected humans regardless of age, sex, or immune status. The mechanism of bone destruction caused by species remained partially unknown due to the lack of a suitable animal model. Here, to study this complication, we explored the suitability of the use of the NOD- mouse to study osteoarticular brucellosis and examined the potential use of this strain to evaluate the safety of live attenuated vaccine candidates. Mice were inoculated intraperitoneally with a single dose of 1 × 10, 1 × 10, or 1 × 10 CFU of S19 or the vaccine candidate S19 and monitored for the development of side effects, including osteoarticular disease, for 13 weeks. Decreased body temperature, weight loss, splenomegaly, and deformation of the tails were observed in mice inoculated with S19 but not in those inoculated with S19 Histologically, all S19-inoculated mice had a severe dose-dependent inflammatory response in multiple organs. The inflammatory response at the tail was characterized by the recruitment of large numbers of neutrophils, macrophages, and osteoclasts with marked bone destruction. These lesions histologically resembled what is typically observed in -infected patients. In contrast, mice inoculated with S19 did not show significant bone changes. Immunofluorescence, hybridization, and confocal imaging demonstrated the presence of at the sites of inflammation, both intra- and extracellularly, and large numbers of bacteria were observed within mature osteoclasts. These results demonstrate the potential use of the NOD- mouse model to evaluate vaccine safety and further study osteoarticular brucellosis.
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http://dx.doi.org/10.1128/IAI.00901-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529653PMC
June 2019

Quantitative Yeast Genetic Interaction Profiling of Bacterial Effector Proteins Uncovers a Role for the Human Retromer in Salmonella Infection.

Cell Syst 2018 09 1;7(3):323-338.e6. Epub 2018 Aug 1.

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77802, USA. Electronic address:

Intracellular bacterial pathogens secrete a repertoire of effector proteins into host cells that are required to hijack cellular pathways and cause disease. Despite decades of research, the molecular functions of most bacterial effectors remain unclear. To address this gap, we generated quantitative genetic interaction profiles between 36 validated and putative effectors from three evolutionarily divergent human bacterial pathogens and 4,190 yeast deletion strains. Correlating effector-generated profiles with those of yeast mutants, we recapitulated known biology for several effectors with remarkable specificity and predicted previously unknown functions for others. Biochemical and functional validation in human cells revealed a role for an uncharacterized component of the Salmonella SPI-2 translocon, SseC, in regulating maintenance of the Salmonella vacuole through interactions with components of the host retromer complex. These results exhibit the power of genetic interaction profiling to discover and dissect complex biology at the host-pathogen interface.
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http://dx.doi.org/10.1016/j.cels.2018.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160342PMC
September 2018

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of .

Front Cell Infect Microbiol 2018 20;8:103. Epub 2018 Apr 20.

Key Laboratory of Zoonosis Research, Ministry of Education, College of Plant Sciences, Jilin University, Changchun, China.

spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to and intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of .
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http://dx.doi.org/10.3389/fcimb.2018.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919948PMC
April 2019

Human Brucellosis and Adverse Pregnancy Outcomes.

Curr Trop Med Rep 2016 Dec 1;3(4):164-172. Epub 2016 Oct 1.

Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA.

Purpose Of Review: Brucellosis is a neglected, zoonotic disease of nearly worldwide distribution. Despite brucellosis being recognized as a reproductive disease in animals, it has been historically known as a flu-like illness in humans with little or no significant role in maternal or newborn health. This review focuses on what is currently known relative to the epidemiology of brucellosis in human pregnancy as well as new insights of placental immunology.

Recent Findings: New evidence suggests that maternal infection poses a significant risk factor for adverse pregnancy outcomes including increased risk for miscarriage during the first and second trimester of gestation, preterm delivery, and vertical transmission to the fetus. Adverse pregnancy outcomes were not associated with any specific clinical sign. However, prompt diagnosis and treatment significantly decreased the risk of miscarriage or any other adverse effect.

Summary: Brucellosis during pregnancy should be considered a significant risk factor for adverse pregnancy outcomes in humans. The identification of the mechanism behind bacterial tropism should prove powerful for the development of new countermeasures to prevent these detrimental effects. Increased awareness concerning brucellosis in pregnant women, its transmission, and prevention measures should be considered as a pressing need.
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http://dx.doi.org/10.1007/s40475-016-0092-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720386PMC
December 2016

Mucosal bacterial dissemination in a rhesus macaque model of experimental brucellosis.

J Med Primatol 2018 02 2;47(1):75-77. Epub 2017 Jun 2.

Tulane National Primate Research Center, Covington, LA, USA.

Animals were experimentally infected with Brucella melitensis via aerosol. B. melitensis was cultured from the saliva and vaginal vault of infected animals, corresponding to bacterial dissemination in other target tissues. This is the first report of bacterial dissemination to these mucosal surfaces in a non-human primate model of brucellosis.
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http://dx.doi.org/10.1111/jmp.12282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946315PMC
February 2018

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection.

Cell Host Microbe 2017 May;21(5):637-649.e6

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, College Station, Texas 77843, USA; Norman Borlaug Center, Texas A&M University, College Station, Texas 77843, USA; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA. Electronic address:

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.
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http://dx.doi.org/10.1016/j.chom.2017.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538893PMC
May 2017

A spp. Isolate from a Pac-Man Frog () Reveals Characteristics Departing from Classical Brucellae.

Front Cell Infect Microbiol 2016 28;6:116. Epub 2016 Sep 28.

Institut National de la Santé et de la Recherche Médicale, U1047, UFR de MédecineNîmes, France; Université de Montpellier, U1047Nîmes, France.

are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog () at a veterinary hospital in Texas and was initially misidentified as . We found that B13-0095 belongs to a group of early-diverging brucellae that includes strain BO1 and the -like strain BO2, with traits that depart significantly from those of the "classical" spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical , but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the genus. Accurate identification tools for such atypical isolates and careful evaluation of their zoonotic potential, are urgently required.
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http://dx.doi.org/10.3389/fcimb.2016.00116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040101PMC
September 2017

The Case for Live Attenuated Vaccines against the Neglected Zoonotic Diseases Brucellosis and Bovine Tuberculosis.

PLoS Negl Trop Dis 2016 08 18;10(8):e0004572. Epub 2016 Aug 18.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, Texas, United States of America.

Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms.
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http://dx.doi.org/10.1371/journal.pntd.0004572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990199PMC
August 2016

Space: A Final Frontier for Vacuolar Pathogens.

Traffic 2016 May 24;17(5):461-74. Epub 2016 Feb 24.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, TX, USA.

There is a fundamental gap in our understanding of how a eukaryotic cell apportions the limited space within its cell membrane. Upon infection, a cell competes with intracellular pathogens for control of this same precious resource. The struggle between pathogen and host provides us with an opportunity to uncover the mechanisms regulating subcellular space by understanding how pathogens modulate vesicular traffic and membrane fusion events to create a specialized compartment for replication. By comparing several important intracellular pathogens, we review the molecular mechanisms and trafficking pathways that drive two space allocation strategies, the formation of tight and spacious pathogen-containing vacuoles. Additionally, we discuss the potential advantages of each pathogenic lifestyle, the broader implications these lifestyles might have for cellular biology and outline exciting opportunities for future investigation.
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http://dx.doi.org/10.1111/tra.12382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048968PMC
May 2016

Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

Am J Pathol 2015 Jun 17;185(6):1505-17. Epub 2015 Apr 17.

Department of Veterinary Pathobiology, Texas A&M University and Texas AgriLife Research, College Station, Texas. Electronic address:

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics.
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http://dx.doi.org/10.1016/j.ajpath.2015.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450313PMC
June 2015

SiRNA screens using Drosophila cells to identify host factors required for infection.

Methods Mol Biol 2014 ;1197:229-44

Department of Veterinary Pathobiology, Texas A&M University, 4474 TAMU, College Station, TX, 77843, USA,

Drosophila melanogaster offers a powerful model system for interrogating interactions between host cells and human bacterial pathogens. Brucella, a gram-negative, facultative intracellular bacterium is the causative agent of brucellosis, a zoonotic disease of global consequence. Over the past several decades, pathogen factors that mediate Brucella infection have been identified. However, host factors that mediate infection have remained obscure. We have used the power of the Drosophila S2 cell system to identify and characterize host factors that support infection by Brucella melitensis. Host protein inositol-requiring enzyme 1 (IRE1α), a transmembrane kinase and master regulator of the eukaryotic unfolded protein response, was shown to play an important role in regulating Brucella infection, thereby providing the first glimpse of host mechanisms that are subverted by the pathogen to support its intracellular lifestyle. Furthermore, our study also established the Drosophila S2 cell as a powerful system for elucidating Brucella host factors. Here, we describe a protocol for using the Drosophila S2 cell system for studying the Brucella-host interaction.
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http://dx.doi.org/10.1007/978-1-4939-1261-2_13DOI Listing
May 2015

Brucella dissociation is essential for macrophage egress and bacterial dissemination.

Front Cell Infect Microbiol 2014 5;4:23. Epub 2014 Mar 5.

Department of Veterinary Pathobiology, Texas A&M University and Texas Agricultural Experiment Station College Station, TX, USA.

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.
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http://dx.doi.org/10.3389/fcimb.2014.00023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942807PMC
September 2014

Aerosol-induced brucellosis increases TLR-2 expression and increased complexity in the microanatomy of astroglia in rhesus macaques.

Front Cell Infect Microbiol 2013 2;3:86. Epub 2013 Dec 2.

Program in Biomedical Science, Tulane School of Medicine New Orleans, LA, USA ; Divisions of Comparative Pathology and Microbiology, Tulane National Primate Research Center Covington, LA, USA ; Department of Microbiology & Immunology, Tulane School of Medicine New Orleans, LA, USA.

Brucella melitensis, a bacterial pathogen and agent of epizootic abortion causes multiple pathologies in humans as well as a number of agriculturally important animal species. Clinical human brucellosis manifests as a non-specific, chronic debilitating disease characterized by undulant fever, arthropathies, cardiomyopathies and neurological sequelae. These symptoms can occur acutely for a few weeks or persist for months to years. Within the brain, endothelial and glial cells can be infected leading to downstream activation events including matrix metalloprotease (MMP) and cytokine secretion and Toll-like receptor (TLR) signaling. These events are likely to lead to tissue remodeling, including morphologic changes in neuronal and glial cells, which are linked to neurological complications including depressive behavior, immune activation and memory loss. Our hypothesis was that B. melitensis infection and neurobrucellosis would lead to activation of astrocytes through upregulation of TLR2 and stimulate concurrent changes in the microanatomy. All six animals were infected via inhalation route. TLR2 expression was approximately doubled in white matter astrocytes of infected rhesus macaques. There was also a 50% increase in the number of astrocytes per unit area in subcortical white matter tracts suggesting increased innate immune activation. This coincided with dramatic increases in the length and complexity of the cell arbor of hypertrophic astrocytes in both cortical gray and white matter. Thus, aerosol-induced brucellosis results in dramatically increased innate immune activation of astrocytes in the absence of widespread neuroinflammation.
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http://dx.doi.org/10.3389/fcimb.2013.00086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844859PMC
April 2014

Host-Brucella interactions and the Brucella genome as tools for subunit antigen discovery and immunization against brucellosis.

Front Cell Infect Microbiol 2013 16;3:17. Epub 2013 May 16.

Department of Veterinary Pathobiology, Texas A&M University College Station, TX 77843, USA.

Vaccination is the most important approach to counteract infectious diseases. Thus, the development of new and improved vaccines for existing, emerging, and re-emerging diseases is an area of great interest to the scientific community and general public. Traditional approaches to subunit antigen discovery and vaccine development lack consideration for the critical aspects of public safety and activation of relevant protective host immunity. The availability of genomic sequences for pathogenic Brucella spp. and their hosts have led to development of systems-wide analytical tools that have provided a better understanding of host and pathogen physiology while also beginning to unravel the intricacies at the host-pathogen interface. Advances in pathogen biology, host immunology, and host-agent interactions have the potential to serve as a platform for the design and implementation of better-targeted antigen discovery approaches. With emphasis on Brucella spp., we probe the biological aspects of host and pathogen that merit consideration in the targeted design of subunit antigen discovery and vaccine development.
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http://dx.doi.org/10.3389/fcimb.2013.00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655278PMC
December 2013

Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach.

PLoS One 2013 22;8(3):e59751. Epub 2013 Mar 22.

Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, United States of America.

Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059751PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606113PMC
September 2013

Diverse genetic regulon of the virulence-associated transcriptional regulator MucR in Brucella abortus 2308.

Infect Immun 2013 Apr 14;81(4):1040-51. Epub 2013 Jan 14.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina, USA.

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants.
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http://dx.doi.org/10.1128/IAI.01097-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639602PMC
April 2013

Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice.

Front Cell Infect Microbiol 2012 7;2:115. Epub 2012 Sep 7.

Department of Veterinary Pathobiology, Texas A&M University, College Station TX, USA.

Brucella spp. cause undulant fever in humans and brucellosis in variety of other animals. Both innate and adaptive immunity have been shown to be important in controlling Brucella infection. Toll-like receptors (TLRs) represent a group of pattern recognition receptors (PRRs) that play critical roles in the host innate immune response, as well as development of adaptive immunity. In the current report, we investigated the role of TLR signaling in the clearance of Brucella and development of adaptive immunity in TLR2(-/-), TLR4(-/-), or MyD88(-/-) mice following aerosol exposure to B. melitensis 16 M. Consistent with previous reports, MyD88 is required for efficient clearance of Brucella from all three organs (lung, spleen, and liver). The results reveal Th2-skewed immune responses in TLR2(-/-) mice late in infection and support a TLR2 requirement for efficient clearance of Brucella from the lungs, but not from the spleen or liver. Similarly, TLR4 is required for efficient clearance of Brucella from the lung, but exhibits a minor contribution to clearance from the spleen and no demonstrable contribution to clearance from the liver. Lymphocyte proliferation assays suggest that the TLRs are not involved in the development of cell-mediated memory response to Brucella antigen. Antibody detection reveals that TLR2 and 4 are required to generate early antigen-specific IgG, but not during the late stages of infection. TLR2 and 4 are only transiently required for IgM production and not at all for IgA production. In contrast, MyD88 is essential for antigen specific IgG production late in infection, but is not required for IgM generation over the course of infection. Surprisingly, despite the prominent role for MyD88 in clearance from all tissues, MyD88-knockout mice express significantly higher levels of serum IgA. These results confirm the important role of MyD88 in controlling infection in the spleen while providing evidence of a prominent contribution to protection in other tissues. In addition, although TLR4 and TLR2 contribute little to control of spleen infection, a significant contribution to clearance of lung infection is described.
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http://dx.doi.org/10.3389/fcimb.2012.00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435510PMC
December 2013

Bacterial Exchange via Nanotubes: Lessons Learned from the History of Molecular Biology.

Authors:
Thomas A Ficht

Front Microbiol 2011 8;2:179. Epub 2011 Sep 8.

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University College Station, TX, USA.

DNA transfer between bacteria has a long and storied history. Starting shortly after the discovery by Avery, MacLeod, and McCarty that DNA was the genetic material, the exchange of DNA between bacteria confirmed that DNA transfer could stably change the phenotypic behavior of organisms. Continued effort along these lines led to the discovery of conjugation systems, bacteriophage transduction, bacterial genome mapping, and to some represents the birth of molecular biology. Recent findings by Dubey and Ben-Yehuda (2011) expand on these early results by suggesting that exchange between bacteria may occur continuously under certain growth conditions via nanotubes. These nanotubes have a structure similar to cell membranes and are sensitive to mild detergent treatment. Transfer of protein and plasmid DNA was demonstrated directly between neighboring and distant bacteria of the same and different genera. Transfer of RNA cannot be ruled out and the transfer of chromosomal DNA was not addressed. This work may reveal an important mechanism behind the spread of antibiotic resistance, however, much work remains to be done in order to confirm or refute the role of this mechanism in the dangerous spread of antibiotic resistance within the prokaryotic biosphere. The work of early molecular biology pioneers can be used as inspiration, if not as a direct template to guide future experimental confirmation.
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http://dx.doi.org/10.3389/fmicb.2011.00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3168790PMC
November 2011

Lipopolysaccharide-Deficient Brucella Variants Arise Spontaneously during Infection.

Front Microbiol 2011 23;2:54. Epub 2011 Mar 23.

Veterinary Pathobiology and Faculty of Genetics, Texas A&M University and Texas AgriLife Research College Station, TX, USA.

Lipopolysaccharide-deficient mutants of smooth Brucella species (rough mutants) have been shown to arise spontaneously in culture. However, in situ analysis of Brucella infected macrophages using antibody directed against O-polysaccharide suggested a loss of reactivity of Brucella consistent with the appearance of rough organisms, and a potential contribution to infection. The experiments reported describe the direct recovery of Brucella from macrophages infected in vitro and from the spleens of infected mice at a frequency similar to that described in vitro, suggesting that Brucella dissociation is not simply an in vitro artifact. The frequency of appearance of spontaneous rough organisms deficient in O-polysaccharide expression measured in vitro is approximately 2-3 logs higher than the appearance of mutation to antibiotic resistance, purine auxotrophy, or reversion of erythritol sensitive ΔeryC mutants to tolerance. Genetic trans-complementation using a plasmid-based expression of Brucella manBA successfully restored O-polysaccharide expression in only one-third of O-polysaccharide deficient spontaneous mutants. Suggesting that the appearance of rough mutants is caused by mutation at more than one locus. In addition, Sanger sequencing of the manBA structural genes detected multiple sequence changes that may explain the observed phenotypic differences. The presence of O-polysaccharide resulted in macrophage and neutrophil infiltration into the peritoneal cavity and systemic distribution of the organism. In contrast, rough organisms are controlled by resident macrophages or by extracellular killing mechanisms and rapidly cleared from this compartment consistent with the inability to cause disease. Loss of O-polysaccharide expression appears to be stochastic giving rise to organisms with biological properties distinct from the parental smooth organism during the course of infection.
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http://dx.doi.org/10.3389/fmicb.2011.00054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153030PMC
November 2011

Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans.

PLoS Pathog 2011 Jun 16;7(6):e1002078. Epub 2011 Jun 16.

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, United States of America.

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.
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http://dx.doi.org/10.1371/journal.ppat.1002078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116820PMC
June 2011

In vitro mutagenesis of Brucella species.

Methods Mol Biol 2010 ;634:15-35

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.

Three major techniques have been employed for broad-range in vitro mutagenesis of Brucella species. Shotgun approaches capable of generating large libraries of randomly inserted transposon mutants include Tn5, mariner (Himar1), and mini-Tn5 signature-tagged mutagenesis. Allelic exchange has also been extensively employed for targeted gene deletion. In general, plasmid and transposon delivery into Brucella has relied upon electroporation; however, conjugation has also been successfully employed. Both approaches have been used to identify critical virulence determinants necessary for disease and intracellular survival of the organism. Perhaps more importantly these approaches have provided an opportunity to develop attenuated vaccine candidates of improved safety and efficacy. Future experiments are designed to identify individual functions that govern the interaction between host and agent and control intracellular trafficking and survival. Toward this goal, this chapter describes current approaches used to mutagenize Brucella spp.
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http://dx.doi.org/10.1007/978-1-60761-652-8_2DOI Listing
December 2010
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