Maintenance of the Shigella sonnei virulence plasmid is dependent on its repertoire and amino acid sequence of toxin:antitoxin systems

Shigella sonnei is a major cause of bacillary dysentery, and of increasing concern due to the spread of multi-drug resistance. S. sonnei harbours pINV, a ∼ 210 kb plasmid that encodes a Type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here we investigated the molecular basis for the emergence of avirulence in S. sonnei , and show that avirulence mainly results from pINV loss, consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin:antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri . We show that introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contribute to pINV loss. Avirulence also results from deletions of T3SS-associated genes on pINV through recombination between insertion sequences (ISs) on the plasmid; these events differ from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrate that TA systems and ISs influence plasmid dynamics and loss in S. sonnei , and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrialising countries, and an emerging multi-drug resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory, through loss of its virulence plasmid (pINV). Here we decipher the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin:antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri . Our findings highlight how subtle differences in plasmids in closely-related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei . This should facilitate research on this bacterium and vaccine development.

Identification of Disease-Specific Single Amino Acid Polymorphisms Using a Simple Random Forest at Protein-level

Background: The number of human genetic variants deposited into publicly available databases has been increasing exponentially. Among these variants, non-synonymous single nucleotide polymorphisms (nsSNPs), also known as single amino acid polymorphisms (SAPs), have been demonstrated to be strongly correlated with phenotypic variations of traits/diseases. Objective: However, the detailed mechanisms governing the disease association of SAPs remain unclear. Thus, further investigation of new attributes and improvement of the prediction becomes more and more urgent since amount of unknown disease-related SAPs need to be investigated. Method: Based on the principle of random forest (RF), we firstly constructed a new effective prediction model for SAPs associated with a particular disease from protein sequences. Four usual sequence signature extractions were separately performed to select the optimal features. Then SAP peptide lengths from 12 to 202 were also optimized. Results: The optimal models achieve higher than 90% accuracy and area under the curve (AUC) of over 0.9 on all 11 external testing datasets. Finally, the good performance on an independent test set with an accuracy higher than 95% proves the superiority of our method. Conclusion: In this paper, based on random forest (RF), we constructed 11 disease-association prediction models for SAPs from the protein sequence level. All models yield prediction accuracy higher than 90% and area under the curve (AUC) more than 0.9. Our method only using the information of protein sequences are more universal than those that depend on some additional information or predictions about the proteins.

Allele-based analysis revealed the critical functions of region 277–297 in the NorA efflux pump of Staphylococcus aureus

Objectives The NorA efflux pump in Staphylococcus aureus mediates resistance to many fluoroquinolone (FQ) antibiotics. Three norA alleles with high sequence similarity are found in various S. aureus strains exhibiting different FQ resistance profiles. This study aimed to elucidate the underlying molecular basis for the varying efflux activity of these three allelic variations. Methods The norA genotypes of 20 S. aureus isolates were analysed. Multiple alignments and conservative analyses were conducted to explore the evolutionary variations. After heterologous expression in Escherichia coli, seven mutants were constructed for MIC tests, efflux activity and conformational change measurements. Results Three NorA alleles were identified that displayed different FQ MICs and varying efflux activity for ethidium bromide, with the NorAII protein showing the strongest activity. A total of 29 single amino acid polymorphisms were identified by conservative analysis within three allelic peptides, with seven sites densely distributed in the 277–297 region. Mutations of these seven residues in NorAII all significantly impaired drug resistance and efflux activity, and three key mutants showed conformational changes in fluorescence resonance energy transfer (FRET) analysis. Conclusions Evolutionary variations of the 277–297 region could be a major explanation for the functional difference of three norA alleles and serve as a potential target for the development of novel NorA inhibitors.

Global distribution of single amino acid polymorphisms in Plasmodium vivax Duffy-binding-like domain and implications for vaccine development efforts

Plasmodium vivax ( Pv ) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in Pv DBL globally, with an emphasis on India as it remains a major contributor to the global Pv malaria burden. Based on 1358 Pv DBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of Pv DBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the Pv DBL subdomain 2 ( α 1– α 6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of Pv DBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in Pv DBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.