PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

IMPORTANCE: Spn is a dangerous human pathogen capable of causing pneumonia and invasive disease. The virulence factor PspA has been studied for nearly four decades with well-established roles in pneumococcal evasion of C-reactive protein and neutralization of lactoferricin. Herein, we show that mammalian (m)GAPDH in mucosal secretions promotes aggregation of pneumococci in a PspA-dependent fashion, whereas lactoferrin counters this effect. PspA-mediated GAPDH-dependent bacterial aggregation protected Spn in nasal lavage elutes and grown in vitro from desiccation on fomites. Furthermore, surviving pneumococci within these aggregates retained their ability to colonize naïve hosts after desiccation. We report that Spn binds to and forms protein complexes on its surface composed of PspA, mGAPDH, and lactoferrin. Changes in the levels of these proteins therefore most likely have critical implications on Spn colonization, survival on fomites, and transmission.

PspA-mediated aggregation protects Streptococcus pneumoniae against desiccation on fomites.

Streptococcus pneumoniae (Spn) resides in the nasopharynx where it can disseminate to cause disease. One key Spn virulence factor is pneumococcal surface protein A (PspA), which promotes survival by blocking the antimicrobial peptide lactoferricin. PspA has also been shown to mediate attachment to dying epithelial cells in the lower airway due to its binding of cell surface-bound mammalian (m)GAPDH. Importantly, the role of PspA during colonization is not well understood. Wildtype Spn was present in nasal lavage elutes collected from asymptomatically colonized mice at levels ~10-fold higher that its isogenic PspA-deficient mutant (ΔpspA). Wildtype Spn also formed aggregates in mucosal secretions composed of sloughed epithelial cells and hundreds of pneumococci, whereas ΔpspA did not. Spn within the center of these aggregates better survived prolonged desiccation on fomites than individual pneumococci and were capable of infecting naïve mice, indicating PspA-mediated aggregation conferred a survival/transmission advantage. Incubation of Spn in saline containing mGAPDH also enhanced tolerance to desiccation, but only for wildtype Spn. mGAPDH was sufficient to cause low-level aggregation of wildtype Spn but not ΔpspA. In strain WU2, the subdomain of PspA responsible for binding GAPDH (aa230-281) is ensconced within the lactoferrin (LF)-binding domain (aa167-288). We observed that LF inhibited GAPDH-mediated aggregation and desiccation tolerance. Using surface plasmon resonance, we determined that Spn forms multimeric complexes of PspA-GAPDH-LF on its surface and that LF dislodges GAPDH. Our findings have important implications regarding pneumococcal colonization/transmission processes and ongoing PspA-focused immunization efforts for this deadly pathogen.

Co-exposure of chromium or cadmium and a low concentration of amoxicillin are responsible to emerge amoxicillin resistant Staphylococcus aureus.

BACKGROUND: Heavy metals and antimicrobials co-exist in many environmental settings. The co-exposure of heavy metals and antimicrobials can drive emergence of antimicrobial resistant (AMR) Enterobacteriaceae. We hypothesized that co-exposure to heavy metals and a low concentration of antibiotic might alter antimicrobial susceptibility patterns, which facilitate emergence of AMR Staphylococcus aureus. METHODS: The growth kinetics of antimicrobial susceptible S. aureus was carried out in the presence of chromium or cadmium salt and a low concentration of antibiotics. Subsequently, the antimicrobial susceptibility pattern was determined by the Kirby-Bauer disc diffusion method. Moreover, the mRNA copy number was determined by reverse transcription polymerase chain reaction. RESULTS: The antimicrobial susceptibility profile revealed that the zone of inhibition (ZOI) for ampicillin, amoxicillin, ciprofloxacin and doxycycline was significantly decreased in chromium pre-exposed S. aureus compared to unexposed bacteria, whereas cadmium pre-exposed bacteria only showed significant decreased in ZOI for amoxicillin. Moreover, the MIC of amoxicillin for S. aureus was increased by 8-fold in chromium and 32-fold in cadmium when bacteria were co-exposed with low concentrations of amoxicillin. The mRNA expression of femX, mepA and norA also significantly increased in S. aureus after exposure to chromium and a low concentration of amoxicillin. CONCLUSION: Cultivation of S. aureus at the minimum levels of chromium or cadmium and a low concentration of amoxicillin increased the inhibitory concentration of amoxicillin through inducing bacterial efflux pumps and antibiotic resistant genes. However, it is warranted to assess the whole transcriptome to find out all responsible factors behind this de novo amoxicillin resistant S. aureus.

Plasmodium falciparum DDX3X is a nucleocytoplasmic protein and requires N-terminal for DNA helicase activity.

Malaria is a haemato-protozoan disease which causes thousands of deaths every year. Due to the alarming increase of drug resistant strains of P. falciparum, malaria is now becoming more deadly. Helicases are the most important components of the cellular machinery without which cells are unable to survive. The importance of helicases has been proven in variety of organisms. In this study we have reported detailed biochemical characterization of human homologue of DDX3X from Plasmodium falciparum (PfDDX3X). Our study revealed that PfDDX3X is ATP- dependent DNA helicase whereas in human host it is ATP-dependent RNA helicase. We show that N-terminal is essential for its activity and it is present in nucleus and cytoplasm in intraerythrocytic developmental stages of P. falciparum 3D7 strain. Also, it is highly expressed in the schizont stage of P. falciparum 3D7strain. The present study suggests that a protein can perform different functions in different systems. The present study will help to understand the basic biology of malaria parasite P. falciparum.

Plasmodium falciparum DDX17 is an RNA helicase crucial for parasite development.

Malaria is one of the major global health concerns still prevailing in this 21st century. Even the effect of artemisinin combination therapies (ACT) have declined and causing more mortality across the globe. Therefore, it is important to understand the basic biology of malaria parasite in order to find novel drug targets. Helicases play important role in nucleic acid metabolism and are components of cellular machinery in various organisms. In this manuscript we have performed the biochemical characterization of homologue of DDX17 from Plasmodium falciparum (PfDDX17). Our results show that PfDDX17 is an active RNA helicase and uses mostly ATP for its function. The qRT-PCR experiment results suggest that PfDDX17 is highly expressed in the trophozoite stage and it is localised mainly in the cytoplasm and in infected RBC (iRBC) membrane mostly in the trophozoite stage. The dsRNA knockdown study suggests that PfDDX17 is important for cell cycle progression. These studies report the biochemical functions of PfDDX17 helicase and further augment the fundamental knowledge about helicase families of P. falciparum.

Plasmodium falciparum DDX55 is a nucleocytoplasmic protein and a 3'-5' direction-specific DNA helicase.

Malaria is one of the major causes of mortality as well as morbidity in many tropical and subtropical countries around the world. Although artemisinin combination therapies (ACTs) are contributing to substantial decline in the worldwide malaria burden, it is becoming vulnerable by the emergence of artemisinin resistance in Plasmodium falciparum leading to clinical failure of ACTs in Southeast Asia. Helicases play important role in nucleic acid metabolic processes and have been also identified as therapeutic drug target for different diseases. Previously, it has been reported that P. falciparum contains a group of DEAD-box family of helicases which are homologous to Has1 family of yeast. Here, we present the characterization of a member of Has1 family (PlasmoDB number PF3D7_1419100) named as PfDDX55. The biochemical characterization of PfDDX55C revealed that it contains both DNA- and RNA-dependent ATPase activity. PfDDX55C unwinds partially duplex DNA in 3' to 5' direction and utilizes mainly ATP or dATP for its activity. The immunofluorescence assay and q-RT PCR analysis show that PfDDX55 is a nucleocytoplasmic protein expressed in all the intraerythrocytic development of P. falciparum 3D7 strain with maximum expression level in trophozoite stage. The LC-MS/MS experiment results and STRING analysis show that PfDDX55 interacts with AAA-ATPase which has been shown to be involved in ribosomal biogenesis.

Plasmodium falciparum DDX31 is DNA helicase localized in nucleolus.

Malaria is a major infectious disease and is responsible for millions of infections every year. As drug resistance strains of Plasmodium species are emerging, there is an urgent need to understand the parasite biology and identify new drug targets. Helicases are very important enzymes that participate in various nucleic acid metabolic processes. Previously we have reported several putative DEAD box helicases in the genome of Plasmodium falciparum 3D7 strain. In this study, we present biochemical characterization of one of the members of Has1 (Helicase associated with SET1) family of DEAD box proteins from P. falciparum 3D7 strain. PfDDX31 is a homologue of human DDX31 helicase and contains all the conserved characteristics motifs. The core PfDDX31C exhibits DNA and RNA dependent ATPase activity and unwinds partially duplex DNA by utilizing ATP or dATP only. The immunofluorescence assay results show that PfDDX31 is expressed throughout all the intraerythrocytic developmental stages in P. falciparum 3D7 strain. The co-localization with nucleolar marker PfNop1 further suggests that PfDDX31 is mostly present in nucleolus, a discrete nuclear compartment.

Biochemical characterization of Plasmodium falciparum parasite specific helicase 1 (PfPSH1).

Malaria, a disease caused by infection with parasites of the genus Plasmodium, causes millions of deaths worldwide annually. Of the five Plasmodium species that can infect humans, Plasmodium falciparum causes the most serious parasitic infection. The emergence of drug resistance and the ineffectiveness of old therapeutic regimes against malaria mean there is an urgent need to better understand the basic biology of the malaria parasite. Previously, we have reported the presence of parasite-specific helicases identified through genome-wide analysis of the P. falciparum (3D7) strain. Helicases are involved in various biological pathways in addition to nucleic acid metabolism, making them an important target of study. Here, we report the detailed biochemical characterization of P. falciparum parasite-specific helicase 1 (PfPSH1) and the effect of phosphorylation on its biochemical activities. The C-terminal of PfPSH1 (PfPSH1C) containing all conserved domains was used for biochemical characterization. PfPSH1C exhibits DNA- or ribonucleic acid (RNA)-stimulated ATPase activity, and it can unwind DNA and RNA duplex substrates. It shows bipolar directionality because it can translocate in both (3'-5' and 5'-3') directions. PfPSH1 is mainly localized to the cytoplasm during early stages (including ring and trophozoite stages of intraerythrocytic development), but at late stages, it is partially located in the cytoplasm. The biochemical activities of PfPSH1 are upregulated after phosphorylation with PKC. The detailed biochemical characterization of PfPSH1 will help us understand its functional role in the parasite and pave the way for future studies.

Novel intergenotype human norovirus recombinant GII.16/GII.3 in Bangladesh.

Noroviruses (NoVs) are one of the major etiological agents of acute gastroenteritis in all age groups. In this study, we identified an intergenotype NoV recombinant strain in the fecal specimens of two male infants with acute diarrhea in Bangladesh. Phylogenetic analysis showed that the identified strains were recombinant NoV strains with a GII.3 capsid and a GII.16 polymerase gene. The recombination breakpoint was located in the ORF1/ORF2 overlap region. To the best of our knowledge this is the first report of a NoV recombinant GII.16/GII.3 strain worldwide.