Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors.

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.

LETHAL TOXIN NEUTRALIZING ANTIBODY RESPONSE INDUCED FOLLOWING ORAL VACCINATION WITH A MICROENCAPSULATED BACILLUS ANTHRACIS STERNE STRAIN 34F2 VACCINE PROOF-OF-CONCEPT STUDY IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

Improved methods are needed to prevent wildlife deaths from anthrax. Caused by Bacillus anthracis, naturally occurring outbreaks of anthrax are frequent but unpredictable. The commercially available veterinary vaccine is labeled for subcutaneous injection and is impractical for large-scale wildlife vaccination programs; therefore, oral vaccination is the most realistic method to control and prevent these outbreaks. We reported the induction of an anthrax-specific lethal toxin (LeTx) neutralizing antibody response in mice following oral vaccination with alginate microcapsules containing B. anthracis Sterne strain 34F2 spores, coated with poly-L-lysine (PLL) and vitelline protein B (VpB). We continued evaluating our novel vaccine formulation through this proof-of-concept study in white-tailed deer (WTD; Odocoileus virginianus; n = 9). We orally vaccinated WTD via needle-free syringe with three formulations of the encapsulated vaccine: 1) PLL-VpB-coated microcapsules with 107-8 spores/ml (n = 5), 2) PLL-VpB-coated microcapsules with 109-10 spores/ml (n = 2), and 3) PLL-coated microcapsules with 109-10 spores/ml (n = 2). Although the limited sample sizes require continued experimentation, we observed an anthrax-specific antibody response in WTD serum following oral vaccination with PLL-coated microcapsules containing 109 spores/ ml. Furthermore, this antibody response neutralized anthrax LeTx in vitro, suggesting that continued development of this vaccine may allow for realistic wildlife anthrax vaccination programs.

In Vitro Protection and Titer Duration of Anthrax-Specific Antibodies Following Subcutaneous Vaccination of White-tailed Deer (Odocoileus virginianus) with Bacillus anthracis Sterne 34F2 Strain Spores.

Outbreaks of anthrax, caused by the soilborne bacterium Bacillus anthracis, are a continuous threat to free-ranging livestock and wildlife in enzootic regions of the United States, sometimes causing mass mortalities. Injectable anthrax vaccines are commercially available for use in livestock, and although hand injection is not a cost- or time-effective long-term management plan for prevention in wildlife, it may provide a tool for managers to target selectively animals of high conservation or economic value. Vaccine-induced anthrax-specific antibody responses have been reported previously in white-tailed deer (Odocoileus virginianus), but the protective nature was not determined. In this study, five white-tailed deer were subcutaneously vaccinated with one dose (1 mL) of the Anthrax Spore Vaccine. Eight blood collections by jugular venipuncture were conducted over 146 d to measure the anthrax-specific antibody response in each deer's serum over time. Antibodies were first detected by ELISA and later with toxin neutralization assays to estimate in vitro protection. Average peak absorbance by ELISA occurred at 14 d postvaccination, whereas average peak in vitro protection occurred at 28 d postvaccination. Observed in vitro protection on average for white-tailed deer after this single-dose vaccination protocol lasted 42-56 d postvaccination, although three individuals still maintained lethal toxin-neutralizing serum antibody titers out to 112 d postvaccination. Vaccination responses were variable but effective to some degree in all white-tailed deer.

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease.

Main protease (M Pro ) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 Cα atom in an M Pro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the M Pro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent M Pro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC 50 value in inhibiting SARS-CoV-2 from infecting ACE2 + A549 cells and a selectivity index of 875. The crystallography analyses of M Pro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent M Pro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 M Pro as therapeutics for COVID-19.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

Case Report: Paucisymptomatic College-Age Population as a Reservoir for Potentially Neutralization-Resistant Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

To better understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant lineage distribution in a college campus population, we carried out viral genome surveillance over a 7-week period from January to March 2021. Among the sequences were three novel viral variants: BV-1 with a B.1.1.7/20I genetic background and an additional spike mutation Q493R, associated with a mild but longer-than-usual COVID-19 case in a college-age person, BV-2 with a T478K mutation on a 20B genetic background, and BV-3, an apparent recombinant lineage. This work highlights the potential of an undervaccinated younger population as a reservoir for the spread and generation of novel variants. This also demonstrates the value of whole genome sequencing as a routine disease surveillance tool.

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

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-log10 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.

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

An oral vaccine against anthrax (Bacillus anthracis) 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 B. anthracis 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 Fasciola hepatica. 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.

Intratracheal Inoculation with Brucella melitensis in the Pregnant Guinea Pig Is an Improved Model for Reproductive Pathogenesis and Vaccine Studies.

Reproductive failure is the hallmark of brucellosis in animals. An uncommon but important complication in pregnant women who become acutely infected with Brucella melitensis is spontaneous pregnancy loss or vertical transmission to the fetus. Unfortunately, the mechanism behind reproductive failure is still obscure, partially due to the lack of a proper study model. Recently, it was demonstrated that intratracheal (IT) inoculation of nonpregnant guinea pigs would replicate features of clinical disease in humans. To determine if IT inoculation would induce reproductive disease, guinea pigs were infected at mid-gestation and monitored daily for fever and abortions. Fever developed between day 14 to 18 postinoculation, and by 3 weeks postinoculation, 75% of pregnant guinea pigs experienced stillbirths or spontaneous abortions mimicking natural disease. Next, to investigate the guinea pig as a model for evaluating vaccine efficacy during pregnancy, nonpregnant guinea pigs were vaccinated with S19, 16MΔvjbR + Quil-A, or 100 µl PBS + Quil-A (as control). Guinea pigs were bred and vaccinated guinea pigs were challenged at mid-gestation with B. melitensis IT inoculation and monitored for fever and abortions. Vaccination with both vaccines prevented fever and protected against abortion. Together, this study indicates that pregnant guinea pigs are an appropriate animal model to study reproductive disease and offer an improved model to evaluate the ability of vaccine candidates to protect against a serious manifestation of disease.

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

As a natural host species for Brucella melitensis, pregnant sheep offer an ideal model to evaluate vaccine candidates for safety. B. melitensis 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 Brucella melitensis 16MΔvjbR, pregnant sheep (n = 6) were vaccinated subcutaneously with 1 × 1010 CFU/ml of 16MΔvjbR or 1 × 109 CFU/ml Rev. 1 at a highly susceptible stage of gestation (approximately 70 days). 16MΔvjbR 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 B. melitensis 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 16MΔvjbR 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 × 107 CFU/ml B. melitensis Protection was similar between Rev. 1 and 16MΔvjbR, with no statistical difference in colonization in the target organs. Overall, the 16MΔvjbR 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.IMPORTANCE Brucellosis is one of the most commonly reported zoonotic disease with a worldwide distribution. Of the 12 Brucella species, Brucella melitensis 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 Brucella melitensis 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 16MΔvjbR Our results indicate that 16MΔvjbR 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.

Evaluation of the Efficacy of the Brucella canis RM6/66 ΔvjbR Vaccine Candidate for Protection against B. canis Infection in Mice.

Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 ΔvjbR strain. Mice vaccinated with a dose of 109 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 ΔvjbR was capable of activating canine dendritic cells in vitro These data demonstrate that the B. canis RM6/66 ΔvjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies.IMPORTANCE Canine brucellosis, caused by Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ΔvjbR vaccine strain capable of protecting mice against challenge.

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortusΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

Nck adapter proteins promote podosome biogenesis facilitating extracellular matrix degradation and cancer invasion.

BACKGROUND: Podosomes are membrane-bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion. METHODS: This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA-MB-231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP)  mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far-western blotting. RESULTS: This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short-hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc-Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far-western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells. CONCLUSIONS: Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy.

Vaccine safety studies of Brucella abortus S19 and S19ΔvjbR in pregnant swine.

Brucellosis in swine is caused by Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis 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 S19ΔvjbR. 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 ±â€¯0.508 × 109 CFU of either S19 or S19ΔvjbR. 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-Brucella 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.

Correction: Characterization of an intratracheal aerosol challenge model of Brucella melitensis in guinea pigs.

[This corrects the article DOI: 10.1371/journal.pone.0212457.].

The NOD-scid IL2rγnull Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular brucellosis is the most common complication in Brucella-infected humans regardless of age, sex, or immune status. The mechanism of bone destruction caused by Brucella 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-scid IL2rγnull 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 × 104, 1 × 105, or 1 × 106 CFU of B. abortus S19 or the vaccine candidate B. abortus S19ΔvjbR 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 B. abortus S19 but not in those inoculated with S19ΔvjbR 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 Brucella-infected patients. In contrast, mice inoculated with B. abortus S19ΔvjbR did not show significant bone changes. Immunofluorescence, in situ hybridization, and confocal imaging demonstrated the presence of Brucella 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-scid IL2rγnull mouse model to evaluate vaccine safety and further study osteoarticular brucellosis.

Characterization of an intratracheal aerosol challenge model of Brucella melitensis in guinea pigs.

B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12-17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies.

Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis.

Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers.

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

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.