Bacteriophage Bioengineering: A Transformative Approach for Targeted Drug Discovery and Beyond.

Bacteriophages, the viruses that infect and replicate within bacteria, have long been recognized as potential therapeutic agents against bacterial infections [...].

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

Association of mprF mutations with cross-resistance to daptomycin and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA).

We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.

Identification and characterization of mutations responsible for the ß-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus.

Staphylococcus aureus strains that are susceptible to the ß-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire ß-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0-256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of ß-lactam resistance in OS-MRSA.

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

Complete Genome Sequence of a Panton-Valentine Leukocidin-Negative Staphylococcus aureus Strain Isolated from a Patient with Pervasive Necrotizing Soft Tissue Infection.

The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.

Corrigendum: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

[This corrects the article DOI: 10.3389/fmicb.2019.02838.].

Complete Genome Sequence of the Methicillin-Resistant Staphylococcus aureus Strain JMUB3031, Isolated from a Patient with Fatal Community-Acquired Pneumonia.

Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.

Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Complete genome sequencing of three human clinical isolates of Staphylococcus caprae reveals virulence factors similar to those of S. epidermidis and S. capitis.

BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

Deletion of Mitochondrial Porin Alleviates Stress Sensitivity in the Yeast Model of Shwachman-Diamond Syndrome.

Shwachman-Diamond syndrome (SDS) is a multi-system disorder characterized by bone marrow failure, pancreatic insufficiency, skeletal abnormalities, and increased risk of leukemic transformation. Most patients with SDS contain mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS), encoding a highly conserved protein that has been implicated in ribosome biogenesis. Emerging evidence also suggests a distinct role of SBDS beyond protein translation. Using the yeast model of SDS, we examined the underlying mechanisms that cause cells lacking Sdo1p, the yeast SBDS ortholog, to exhibit reduced tolerance to various stress conditions. Our analysis indicates that the environmental stress response (ESR), heat shock response (HSR), and endoplasmic reticulum unfolded protein response (UPR) of sdo1Δ cells are functional and that defects in these pathways do not produce the phenotypes observed in sdo1Δ yeast. Depletion of mitochondrial DNA (mtDNA) was observed in sdo1Δ cells, and this is a probable cause of the mitochondrial insufficiency in SDS. Prior disruption of POR1, encoding the mitochondrial voltage dependent anion channel (VDAC), abrogated the effects of SDO1 deletion and substantially restored resistance to environmental stressors and protected against damage to mtDNA. Conversely, wild-type cells over-expressing POR1 exhibited growth impairment and increased stress sensitivity similar to that seen in sdo1Δ cells. Overall, our results suggest that specific VDAC inhibitors may have therapeutic benefits for SDS patients.

Improper protein trafficking contributes to artemisinin sensitivity in cells lacking the KDAC Rpd3p.

Lysine deacetylases (KDACs) inhibitors may have therapeutic value in anti-malarial combination therapies with artemisinin. To evaluate connections between KDACs and artemisinin, Saccharomyces cerevisiae deletion mutants in KDAC genes were assayed. Deletion of RPD3, but not other KDAC genes, resulted in strong sensitivity to artemisinin, which was also observed in sit4Δ mutants with impaired endoplasmic reticulum (ER) to Golgi protein trafficking. Decreased accumulation of the transporters Pdr5p, Fur4p, and Tat2p was observed in rpd3Δ and sit4Δ cells. The unfolded protein response is induced in rpd3Δ cells consistent with retention of proteins in the ER. Disruption of protein trafficking appears to sensitize cells to artemisinin and targeting these pathways may be useful as part of artemisinin based anti-malarial therapy.