Titan Cells and Yeast Forms of Cryptococcus neoformans and Cryptococcus gattii Are Recognized by GXMR-CAR

Cryptococcosis, a systemic mycosis that affects both the immunocompromised and immunocompetent, is caused by the inhalation of dehydrated yeasts or fungal spores of Cryptococcus gattii or Cryptococcus neoformans. The Cryptococcus spp. polysaccharide capsule is composed mainly of glucuronoxylomannan—GXM, its major virulence factor. The capsule thickness increases to more than 15 μm during titanization, favoring the pathogenesis of cryptococcosis. Previous studies demonstrated that cytotoxic T cells that had been bioengineered with GXM-targeting chimeric antigen receptor (GXMR-CAR) were able to recognize C. neoformans by promoting the control of titanization. GXMR-CAR, a second-generation CAR, contains a single-chain variable fragment that originates from a 18B7 clone: a human IgG4 hinge, followed by a human CD28 (transmembrane/cytoplasmic domains) and a CD3ς chain. In the current study, we redirected T cells to target distinct C. neoformans and C. gattii cell types by GXMR-CAR. Lentiviral particles carrying the GXMR-CAR sequence were used to transduce Jurkat cells, and these modified cells interacted with the GXM of the C. gattii R265 strain. Moreover, GXMR-CAR mediated the recognition of C. gattii and C. neoformans yeasts with both thin and thick polysaccharide capsules, and GXMR-CAR Jurkat cells interacted with titan cells sourced from both Cryptococcus spp. Thus, bioengineered cells using CAR can improve the treatment of cryptococcosis.

Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus

CRISPR-associated Cas9 endonuclease (CRISPR/Cas9) systems are widely used to introduce precise mutations, such as knocking in/out at targeted genomic sites. Herein, we successfully disrupted the transcription of multiple genes in Bacillus pumilus LG3145 using a series of unspecific guide RNAs (gRNAs) and UgRNA:Cas9 system-assisted cre-box editing. The bases used as gRNAs shared 30–70% similarity with a consensus sequence, a cis-acting element (cre-box) mediating carbon catabolite repression (CCR) of many genes in Bacillus. This triggers trans-crRNA:Cas9 complex wobble cleavage up/downstream of cre sites in the promoters of multiple genes (up to 7), as confirmed by Sanger sequencing and next-generation sequencing (NGS). LG3145 displayed an obvious CCR release phenotype, including numerous secondary metabolites released into the culture broth, ∼ 1.67 g/L white flocculent protein, pigment overflow causing orange-coloured broth (absorbance = 309 nm), polysaccharide capsules appearing outside cells, improved sugar tolerance, and a two-fold increase in cell density. We assessed the relationship between carbon catabolite pathways and phenotype changes caused by unspecific UgRNA-directed cre site wobble editing. We propose a novel strategy for editing consensus targets at operator sequences that mediates transcriptional regulation in bacteria.

RNA thermosensors facilitate Streptococcus pneumoniae and Haemophilus influenzae immune evasion

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted respiratory pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases, remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signals to pathogens. Here, we investigate whether these respiratory pathogens can sense environmental temperature to evade host complement-mediated killing. We show that productions of two vital virulence factors and vaccine components, the polysaccharide capsules and factor H binding proteins, are temperature dependent, thus influencing serum/opsonophagocytic killing of the bacteria. We identify and characterise four novel RNA thermosensors in S. pneumoniae and H. influenzae, responsible for capsular biosynthesis and production of factor H binding proteins. Our data suggest that these bacteria might have independently co-evolved thermosensing abilities with different RNA sequences but distinct secondary structures to evade the immune system.

Uji pigmen dan deteksi kapsul polisakarida pada Staphylococcus aureus isolat asal broiler PIGMENT TEST AND DETECTION OF POLISAKARIDA CAPSULES ON Staphylococcus aureus ISOLAT BROILER

Staphylococcus aureus is a pathogenic bacterium causing disease in humans and animals. In broilers it cause septicemia, tendosinovitis, dermatitis, endocarditis, wound infections and arthritis and bumblefoot. The ability of Staphylococcus aureus to cause disease depends on the virulence factors they bear. The purpose of this research is to investigate the distribution of pigment production type and the existence of genes of polysaccharide capsule phenotype and genotype as determinant factor of virulence of bacteria on 15 isolates of Staphylococcus aureus from broiler. Pigment production test showed that 86.7% of isolates producing yellow pigment and 13.3% isolates produce orange pigment. The detection of polysaccharide capsules was phenotypically performed with hydrophobicity test with serum soft agar medium (SSA) showed 53.3% isolate grow compact and 46,7% isolate grown difuse and hydrophobicity test by salt aggregation test method (SAT) showed 66,7% hydrophil and 33.3% are hydrophobic. Genotype detection of polysaccharide capsule genes by polymerase chain reaction (PCR) showed 66.7% detected cap5 (amplicon 361bp) and 33.3% detected cap8 (173bp ampliole). The type of pigment production and the presence of polysaccharide capsules are some of the virulent factors in Staphylococcus aureus.

Investigating the biomechanical properties of streptococcal polysaccharide capsules using atomic force microscopy

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule, which facilitates immune evasion and is a key virulence determinant. However, recent data has shown that the closely related Streptococcus mitis can also express polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We have used atomic force microscopy (AFM) techniques to investigate the biophysical properties of S. mitis and S. pneumoniae strains expressing the same capsular serotypes that might relate to their differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes S. mitis strains were more susceptible to neutrophil killing and imaging using electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the centre and edge areas of encapsulated S. pneumoniae and S. mitis strains. This “edge effect” was not observed in the unencapsulated streptococcal strains and in an unencapsulated Staphylococcus aureus strain, and therefore was a direct representation of the mechanical properties of the bacterial capsule. When two strains of S. mitis and S. pneumoniae expressed an identical capsular serotype, they presented also similar biomechanical characteristics. This would infer a potential relationship between capsule biochemistry and nanomechanics, independent of the bacterial strains. Overall, AFM was an effective tool to explore the biophysical properties of bacterial capsules of living bacteria by reproducibly quantifying the elastic and adhesive properties of bacterial cell surfaces. Using AFM to investigate capsule differences over a wider range of strains and capsular serotypes of streptococci and correlate the data with phenotypic differences will elucidate how the biophysical properties of the capsule can influence its biological role during infection.

Designing ecologically-optimised vaccines using population genomics

Streptococcus pneumoniae (the pneumococcus) is a common nasopharyngeal commensal capable of infecting normally sterile anatomical sites, resulting in invasive pneumococcal disease (IPD). Effective vaccines preventing IPD exist, but each of the antigens they contain typically induces protective immunity against only one of the approximately 100 pneumococcal serotypes, which are differentiated by immunogenically-distinct polysaccharide capsules. Serotypes vary in their propensity to cause IPD, quantified as their invasiveness. Vaccines are designed to include serotypes commonly isolated from IPD, but the immunity they induce is sufficiently strong to also eliminate vaccine serotypes from carriage. This enables their replacement by non-vaccine serotypes in the nasopharynx. The emergence of invasive non-vaccine serotypes has undermined some vaccination programmes’ benefits. Recent advances in genomics and modeling have enabled forecasting of which non-vaccine serotypes will be successful post-vaccination. Here, we demonstrate that vaccines optimised using this framework can minimise IPD and antibiotic-resistant disease more effectively than existing formulations in the model, through mitigating the consequences of serotype replacement. The simulations also demonstrate that tailoring vaccines to the pre-vaccine bacterial population is likely to have a substantial impact on reducing IPD, highlighting the importance of epidemiological data, genomics and ecological models as tools for vaccine design and evaluation.