Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis.

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38-38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

Canine Staphylococcaceae circulating in a Kenyan animal shelter.

Animal shelters, especially in resource-poor countries, bring together pets from different regions and with different backgrounds. The crowding of such animals often results in infectious diseases, such as respiratory infections. This study characterized Staphylococcaceae from diseased and apparently healthy dogs housed in an animal shelter in Kenya, to determine their antibiotic resistance profiles, their genetic relatedness, and the presence of dominant clones. Therefore, bacteria were collected from all 167 dogs present in the shelter in June 2015 and screened for Staphylococcaceae using standard cultivation techniques. In all, 92 strains were isolated from 85 dogs and subsequently sequenced by PacBio long-read sequencing. Strains encompassed nine validated species, while S. aureus (n = 47), S. pseudintermedius (n = 21), and Mammaliicoccus (M.) sciuri (n = 16) were the three most dominant species. Two S. aureus clones of ST15 (CC15) and ST1292 (CC1) were isolated from 7 and 37 dogs, respectively. All 92 strains isolated were tested for their antimicrobial susceptibility by determining the minimum inhibitory concentrations. In all, 86 strains had resistance-associated minimal inhibitory concentrations to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, kanamycin/gentamicin, or streptomycin. Many virulence-encoding genes were detected in the S. aureus strains, other Staphylococcaceae contained a different set of homologs of such genes. The presence of mobile genetic elements, such as plasmids and prophages, known to facilitate the dissemination of virulence- and resistance-encoding genes, was also assessed. The unsuspected high presence of two S. aureus clones in about 50% of dogs suggests dissemination within the shelter and a human source.IMPORTANCEMicrobiological data from sub-Saharan Africa are scarce compared to data from North America, Europe, or Asia, and data derived from dogs, the man's best friend, kept in sub-Saharan Africa are largely missing. This work presents data on Staphylococcaceae mainly isolated from the nasal cavity of dogs stationed at a Kenyan shelter in 2015. We characterized 92 strains isolated from 85 dogs, diseased and apparently healthy ones. The strains isolated covered nine validated species and we determined their phenotypic resistance and characterized their complete genomes. Interestingly, Staphylococcus aureus of two predominant genetic lineages, likely to be acquired from humans, colonized many dogs. We also detected 15 novel sequence types of Mammaliicoccus sciuri and S. pseudintermedius indicating sub-Saharan-specific phylogenetic lineages. The data presented are baseline data that guide antimicrobial treatment for dogs in the region.

Corrigendum: Evidence for the cytoplasmic localization of the L-α-Glycerophosphate oxidase in members of the "Mycoplasma mycoides cluster".

[This corrects the article DOI: 10.3389/fmicb.2019.01344.].

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136.

Impaired immune response drives age-dependent severity of COVID-19.

Severity of COVID-19 shows an extraordinary correlation with increasing age. We generated a mouse model for severe COVID-19 and show that the age-dependent disease severity is caused by the disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) immunity. Aggravated disease in aged mice was characterized by a diminished IFN-γ response and excessive virus replication. Accordingly, adult IFN-γ receptor-deficient mice phenocopied the age-related disease severity, and supplementation of IFN-γ reversed the increased disease susceptibility of aged mice. Further, we show that therapeutic treatment with IFN-λ in adults and a combinatorial treatment with IFN-γ and IFN-λ in aged Ifnar1-/- mice was highly efficient in protecting against severe disease. Our findings provide an explanation for the age-dependent disease severity and clarify the nonredundant antiviral functions of type I, II, and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Our data suggest that highly vulnerable individuals could benefit from immunotherapy combining IFN-γ and IFN-λ.

The XadA Trimeric Autotransporter Adhesins in Xylella fastidiosa Differentially Contribute to Cell Aggregation, Biofilm Formation, Insect Transmission and Virulence to Plants.

Surface adhesion strategies are widely employed by bacterial pathogens during establishment and systemic spread in their host. A variety of cell-surface appendages such as pili, fimbriae, and afimbrial adhesins are involved in these processes. The phytopathogen Xylella fastidiosa employs several of these structures for efficient colonization of its insect and plant hosts. Among the adhesins encoded in the X. fastidiosa genome, three afimbrial adhesins, XadA1, Hsf/XadA2, and XadA3, are predicted to be trimeric autotransporters with a C-terminal YadA-anchor membrane domain. We analyzed the individual contributions of XadA1, XadA2, and XadA3 to various cellular behaviors both in vitro and in vivo. Using isogenic X. fastidiosa mutants, we found that cell-cell aggregation and biofilm formation were severely impaired in the absence of XadA3. No significant reduction of cell-surface attachment was found with any mutant under flow conditions. Acquisition by insect vectors and transmission to grapevines were reduced in the XadA3 deletion mutant. While the XadA3 mutant was hypervirulent in grapevines, XadA1 or XadA2 deletion mutants conferred lower disease severity than the wild-type strain. This insight of the importance of these adhesive proteins and their individual contributions to different aspects of X. fastidiosa biology should guide new approaches to reduce pathogen transmission and disease development. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Engineering of the Fast-Growing Mycoplasma feriruminatoris toward a Live Vaccine Chassis.

Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the M. feriruminatoris chassis confirmed the corresponding loss of H2O2 production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent Mycoplasma capricolum subsp. capripneumoniae was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems.

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications.

In-yeast reconstruction of the African swine fever virus genome isolated from clinical samples.

This protocol describes a synthetic genomics pipeline to clone and engineer the entire 190-kbp genome of the African swine fever virus (ASFV) genotype II in yeast using transformation-associated recombination cloning. The viral genome was cloned using DNA directly extracted from a clinical sample. In addition, the precise deletion of a non-essential gene and its replacement by a synthetic reporter gene cassette are presented. This protocol is applicable to other ASFV genotypes and other large DNA viruses.

Complete Genome Sequences of the Methicillin-Resistant Strain Staphylococcus aureus 17Gst354 and Its Prophage Staphylococcus Phage vB_StaphS-IVBph354.

We report the complete 2,783,931-bp circular genome sequence of the human methicillin-resistant strain Staphylococcus aureus 17Gst354, isolated from a nasal swab. The strain possessed an additional 4,397-bp plasmid. Moreover, we induced and sequenced its temperate phage Staphylococcus phage vB_StaphS-IVBph354, which has a circular genome of 41,970 bp.

Development of safe and highly protective live-attenuated SARS-CoV-2 vaccine candidates by genome recoding.

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We construct a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and assess their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses do not cause clinical symptoms but are highly immunogenic and induce strong protective immunity. Attenuated viruses replicate efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters develop no signs of disease and rapidly clear challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic.

Establishment of caprine airway epithelial cells grown in an air-liquid interface system to study caprine respiratory viruses and bacteria.

Respiratory diseases negatively impact the global goat industry, but are understudied. There is a shortage of established and biological relevant in vitro or ex vivo assays to study caprine respiratory infections. Here, we describe the establishment of an in vitro system based on well-differentiated caprine airway epithelial cell (AEC) cultures grown under air liquid interface conditions as an experimental platform to study caprine respiratory pathogens. The functional differentiation of the AEC cultures was monitored and confirmed by light and immunofluorescence microscopy, scanning electron microscopy and examination of histological sections. We validated the functionality of the platform by studying Influenza D Virus (IDV) infection and Mycoplasma mycoides subsp. capri (Mmc) colonization over 5 days, including monitoring of infectious agents by titration and qPCR as well as colour changing units, respectively. The inoculation of caprine AEC cultures with IDV showed that efficient viral replication takes place, and revealed that IDV has a marked cell tropism for ciliated cells. Furthermore, AEC cultures were successfully infected with Mmc using a multiplicity of infection of 0.1 and colonization was monitored over several days. Altogether, these results demonstrate that our newly-established caprine AEC cultures can be used to investigate host-pathogen interactions of caprine respiratory pathogens.

SARS-CoV-2 spike D614G change enhances replication and transmission.

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.

SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic 1 . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline.

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

Contagious Bovine and Caprine Pleuropneumonia: a research community's recommendations for the development of better vaccines.

Contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP) are major infectious diseases of ruminants caused by mycoplasmas in Africa and Asia. In contrast with the limited pathology in the respiratory tract of humans infected with mycoplasmas, CBPP and CCPP are devastating diseases associated with high morbidity and mortality. Beyond their obvious impact on animal health, CBPP and CCPP negatively impact the livelihood and wellbeing of a substantial proportion of livestock-dependent people affecting their culture, economy, trade and nutrition. The causative agents of CBPP and CCPP are Mycoplasma mycoides subspecies mycoides and Mycoplasma capricolum subspecies capripneumoniae, respectively, which have been eradicated in most of the developed world. The current vaccines used for disease control consist of a live attenuated CBPP vaccine and a bacterin vaccine for CCPP, which were developed in the 1960s and 1980s, respectively. Both of these vaccines have many limitations, so better vaccines are urgently needed to improve disease control. In this article the research community prioritized biomedical research needs related to challenge models, rational vaccine design and protective immune responses. Therefore, we scrutinized the current vaccines as well as the challenge-, pathogenicity- and immunity models. We highlight research gaps and provide recommendations towards developing safer and more efficacious vaccines against CBPP and CCPP.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

Complete Genome Sequence of Mycoplasma feriruminatoris Strain IVB14/OD_0535, Isolated from an Alpine Ibex in a Swiss Zoo.

Mycoplasma feriruminatoris is a fast-growing and genetically tractable mycoplasma species. We sequenced the Swiss strain IVB14/OD_0535, isolated from an Alpine ibex. This strain has a circular genome of 1,027,435 bp with a G+C content of 24.3%. It encodes 835 open reading frames (ORFs), 2 rRNA operons, and 30 tRNAs.