Distinct Contribution of the HtrA Protease and PDZ Domains to Its Function in Stress Resilience and Virulence of Bacillus anthracis

Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium Bacillus anthracis. We previously demonstrated that disruption of htrA gene, encoding the chaperone/protease HtrABA (High Temperature Requirement A of B. anthracis) results in significant virulence attenuation, despite unaffected ability of ΔhtrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis ΔhtrA strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the htrA gene was suggested to reflect the susceptibility of ΔhtrA mutated strains to stress insults encountered in the host indicating that HtrABA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrABA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrABA. By inspecting the phenotype exhibited by ΔhtrA strains trans-complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrABA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrABA is essential for its N-terminal autolysis and subsequent release into the extracellular milieu, while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrABA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrABA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrABA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrABA amongst bacterial serine proteases of the HtrA family

A systematic view on influenza induced host shutoff

DNA nanotechnology is an emerging field which utilizes the unique structural properties of nucleic acids in order to build nanoscale devices, such as logic gates, motors, walkers, and algorithmic structures. Predicting the structure and interactions of a DNA device requires effective modeling of both the thermodynamics and the kinetics of the DNA strands within the system. The kinetics of a set of DNA strands can be modeled as a continuous time Markov process through the state space of all secondary structures. The primary means of exploring the kinetics of a DNA system is by simulating trajectories through the state space and aggregating data over many such trajectories. We expand on previous work by extending the thermodynamics and kinetics models to handle multiple strands in a fixed volume, in a way that is consistent with previous models. We developed data structures and algorithms that allow us to take advantage of local properties of secondary structure, improving the efficiency of the simulator so that we can handle reasonably large systems. Finally, we illustrate the simulator’s analysis methods on a simple case study

Early Diagnosis of Pathogen Infection by Cell-Based Activation Immunoassay

Diagnostic identification of pathogens is usually accomplished by isolation of the pathogen or its substances, and should correlate with the time and site of infection. Alternatively, immunoassays such as enzyme-linked immunosorbent assay (ELISA) tests for quantification of serum antibodies are expedient and are usually employed for retrospective diagnostic of a particular infective agent. Here, the potential of cell-based immunoassays for early pathogen detection was evaluated by quantification of specific, antigen-activated, low-frequency IFNγ-secreting cells in mouse spleens following infection with various pathogens. Using enzyme-linked immunospot (ELISPOT) assays, specific responses were observed within 3–6 days following infection with F. tularensis, B. anthracis, Y. pestis, or Influenza virus. Blood samples collected from F. tularensis-infected mice revealed the presence of IFNγ-producing activated cells within one week post infection. When non-human primates were infected with B. anthracis, cellular response was observed in peripheral blood samples as early as five days post infection, 3–5 days earlier than serum antibodies. Finally, the expression pattern of genes in splenocytes of F. tularensis-infected mice was inspected by a transcriptomic approach, enabling the identification of potential host targets for the future development of genetic-based cellular immunoassays. Altogether, the data demonstrate the potential of cell-based immunoassays for early pathogen detection

CD38 Facilitates Recovery from Traumatic Brain Injury

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. It causes progressive tissue atrophy and consequent neurological dysfunctions. TBI is accompanied by neuroinflammation, a process mediated largely by microglia. CD38 is an ectoenzyme that promotes transmembrane signaling via the synthesis of potent calcium mobilizing agents or via its receptor activity. CD38 is expressed in the brain in various cell types including microglia. In previous studies, we showed that CD38 regulates microglial activation and response to chemokines. In view of the important role of neuroinflammation in TBI and the effects of CD38 on microglial responses, the present study examines the role of CD38 in the recovery of mice from closed head injury (CHI), a model of focal TBI. For this purpose, CD38-deficient and wild-type (WT) mice were subjected to a similar severity of CHI and the effect of the injury on neurobehavioral and cognitive functions was assessed by the Neurological Severity Score (NSS) and the Object Recognition Test, at various time points post-injury. The results show that recovery after CHI (as indicated by the NSS) was significantly lower in CD38-deficient mice than in WT mice and that the object recognition performance after injury was significantly impaired in injured CD38-deficient mice than in WT mice. In addition, we also observed that the amount of activated microglia/macrophages at the injury site was significantly lower in CD38-deficient mice compared with WT mice. Taken together, our findings indicate that CD38 plays a beneficial role in the recovery of mice from CHI and that this effect is mediated, at least in part, via the effect of CD38 on microglia responses

A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

Here, Noy-Porat, Makdasi et al. report the isolation of a panel of neutralizing mAbs selected against SARS-CoV-2 receptor-binding domain (RBD) from a phage display library constructed based on patient samples collected in the acute phase of the disease, which show efficient neutralizing activities against authentic virus in vitro