Proteomic analysis of Giardia lamblia and Trichomonas vaginalis flagella reveal unique post-translational modifications in tubulin that provide clues to regulation of their motilities.

All eukaryotic flagella are made of microtubules and driven by dynein motor proteins. However, every organism is unique in terms of its flagellar waveform, beat frequency, and its general motility pattern. With recent research, it is becoming clear that despite overall conservation in flagellar structure, the pattern of tubulin post-translational modifications within the flagella are diverse and may contribute to variations in their patterns of motility. In this study, we have analyzed the tubulin post-translational modification in the protozoan parasites Giardia lamblia and Trichomonas vaginalis using global, untargeted mass spectrometry. We show that tubulin monoglycylation is a modification localized to the flagella present in G. lamblia but absent in T. vaginalis. We also show the presence of glutamylated tubulin in both G. lamblia and T. vaginalis. Using MS/MS, we were also able to identify the previously unknown sites of monoglycylation in ß-tubulin at E438 and E439 in G. lamblia. Using isolated flagella, we also characterized the flagellar proteome in G. lamblia and T. vaginalis and identified 475 proteins in G. lamblia and 386 proteins in T. vaginalis flagella. Altogether, the flagellar proteomes as well as the sites of tubulin PTMs in these organisms, reveal potential mechanisms in regulating flagellar motilities in these neglected protozoan parasites.

Proteo-Genomic Analysis of SARS-CoV-2: A Clinical Landscape of Single-Nucleotide Polymorphisms, COVID-19 Proteome, and Host Responses.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next-generation sequencing (NGS) and mass spectrometry on nasopharyngeal swabs of COVID-19 patients to examine the clinical genome and proteome. Our study confirms the mutability of SARS-CoV-2 showing multiple single-nucleotide polymorphisms. NGS analysis detected 27 mutations, of which 14 are synonymous, 11 are missense, and 2 are extragenic in nature. Phylogenetic analysis of SARS-CoV-2 isolates indicated their close relation to a Bangladesh isolate and multiple origins of isolates within the country. Our proteomic analysis, for the first time, identified 13 different SARS-CoV-2 proteins from the clinical swabs. Of the total 41 peptides captured by high-resolution mass spectrometry, 8 matched to nucleocapsid protein, 2 to ORF9b, and 1 to spike glycoprotein and ORF3a, with remaining peptides mapping to ORF1ab polyprotein. Additionally, host proteome analysis revealed several key host proteins to be uniquely expressed in COVID-19 patients. Pathway analysis of these proteins points toward modulation in immune response, especially involving neutrophil and IL-12-mediated signaling. Besides revealing the aspects of host-virus pathogenesis, our study opens new avenues to develop better diagnostic markers and therapeutic approaches.

A complementary forensic 'proteo-genomic' approach for the direct identification of biological fluid traces under fingernails.

Violent contact between individuals during a crime can result in body fluids becoming trapped under the fingernails of the individuals involved. The traces under fingernails represent valuable forensic evidence because DNA profiling can indicate from whom the trace originated and proteomic methods can be used to determine the type of fluid in the trace, thus providing evidence as to the circumstances surrounding the crime. Here, we present an initial study of an analytical strategy that involves two complementary techniques, direct PCR DNA profiling and direct mass spectrometry-based protein biomarker detection, for the comprehensive examination of traces of biological fluids gathered from underneath fingernails. With regard to protein biomarker detection, direct MALDI-ToF MS/MS is very sensitive, allowing results to be obtained from biological material present on only a few fibres plucked from a microswab used to collect the traces. Human cornulin, a protein biomarker for vaginal fluid, could be detected up to 5 h after it had been deposited under fingernails whereas haemoglobin, a biomarker for blood, is somewhat more persistent under fingernails and could be detected up to 18 h post-deposition. Bottom-up tandem mass spectrometry techniques were used to provide a high level of confidence in assigning the identity of protein biomarkers. nLC-ESI-qToF MS/MS offered higher levels of confidence and the ability to detect traces that had been present under fingernails for longer periods of time, but this performance came with the cost of longer analysis time and a more laborious sampling approach. Graphical abstract ᅟ.

A mass spectrometry-based forensic toolbox for imaging and detecting biological fluid evidence in finger marks and fingernail scrapings.

During a crime, biological material such as blood or vaginal fluid may become smeared on the fingers of the victim or suspect or trapped under their fingernails. The type of trapped fluid is extremely valuable forensic information. Furthermore, if either person touches an object at the crime scene with their 'contaminated' finger then a 'contaminated' finger mark may be deposited. Such marks have great value as they could identify not only who deposited the mark but also who they touched and which part of the body they touched. Here, we describe preliminary work towards a 'toolbox' of techniques based on mass spectrometry (MS) for the identification of biological fluid traces under fingernails or the imaging of them in finger marks. Liquid chromatography-multidimensional MS was effective for the detection of protein biomarkers characteristic of vaginal fluid and blood trapped under fingernails, even after hands had been washed. In regard to examination of finger marks for the presence of biological fluids, the most practical implementation of any technique is to integrate it with, but after, routine crime scene finger mark enhancement has been applied. Here, we demonstrate the usage of matrix-assisted laser desorption ionization-time of flight-MS for the detection and mapping of proteins and peptides from body fluids in finger marks, including marks enhanced using aluminium-containing magnetic powder and then 'lifted' with adhesive tape. Hitherto, only small molecules have been detected in enhanced, lifted marks. In a novel development, aluminium in the enhancement powder assisted ionization of small molecules in finger marks to the extent that conventional matrix was not required for MS.

Autophosphorylation of Ser428 of EhC2PK plays a critical role in regulating erythrophagocytosis in the parasite Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn(2+)-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.

Absence of proline-peptide transporter YjiY in Salmonella Typhimurium leads to secretion of factors which inhibits intra-species biofilm formation.

Salmonella is a genus of widely spread Gram negative, facultative anaerobic bacteria, which is known to cause »th of diarrheal morbidity and mortality globally. It causes typhoid fever and gastroenteritis by gaining access to the host gut through contaminated food and water. Salmonella utilizes its biofilm lifestyle to strongly resist antibiotics and persist in the host. Although biofilm removal or dispersal has been studied widely, the inhibition of the initiation of Salmonella Typhimurium (STM WT) biofilm remains elusive. This study demonstrates the anti-biofilm property of the cell-free supernatant obtained from a carbon-starvation induced proline peptide transporter mutant (STM ΔyjiY) strain. The STM ΔyjiY culture supernatant primarily inhibits biofilm initiation by regulating biofilm-associated transcriptional network that is reversed upon complementation (STM ΔyjiY:yjiY). We demonstrate that abundance of FlgM correlates with the absence of flagella in the STM ΔyjiY supernatant treated WT cells. NusG works synergistically with the global transcriptional regulator H-NS. Relatively low abundances of flavoredoxin, glutaredoxin, and thiol peroxidase might lead to accumulation of ROS within the biofilm, and subsequent toxicity in STM ΔyjiY supernatant. This work further suggests that targeting these oxidative stress relieving proteins might be a good choice to reduce Salmonella biofilm.