Structural proteomics, electron cryo-microscopy and structural modeling approaches in bacteria-human protein interactions

A central challenge in infection medicine is to determine the structure and function of host-pathogen protein-protein interactions to understand how these interactions facilitate bacterial adhesion, dissemination and survival. In this review, we focus on proteomics, electron cryo-microscopy and structural modeling to showcase instances where affinity-purification (AP) and cross-linking (XL) mass spectrometry (MS) has advanced our understanding of host-pathogen interactions. We highlight cases where XL-MS in combination with structural modeling has provided insight into the quaternary structure of interspecies protein complexes. We further exemplify how electron cryo-tomography has been used to visualize bacterial-human interactions during attachment and infection. Lastly, we discuss how AP-MS, XL-MS and electron cryo-microscopy and -tomography together with structural modeling approaches can be used in future studies to broaden our knowledge regarding the function, dynamics and evolution of such interactions. This knowledge will be of relevance for future drug and vaccine development programs

Distinct Serotypes of Streptococcal M Proteins Mediate Fibrinogen-Dependent Platelet Activation and Proinflammatory Effects

Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections

Rad26, the Transcription-Coupled Repair Factor in Yeast, Is Required for Removal of Stalled RNA Polymerase-II following UV Irradiation

Transcription coupled nucleotide excision repair (TCR) is a major pathway responsible for removal of helix distorting DNA lesions from transcriptionally active regions of the genome. Rad26, a key factor of the TCR pathway, is known to play a role during early steps of TCR. Here, we show that Rad26-mediated TCR is not absolutely dependent on active transcription elongation in budding yeast. As per our results, RAD26- deleted cells show enhanced UV sensitivity compared to wild type cells under conditions where transcription elongation is inhibited. The increased UV sensitivity observed in RAD26 -deleted cells, however, is not due to reduced expression of the major NER-responsive genes. Interestingly, transcription of the constitutively expressed RPB2 gene is adversely affected in RAD26- deleted cells during UV-induced DNA damage repair. In consonance, chromatin immunoprecipitation analysis showed that unlike in wild type, in RAD26 -deleted cells no significant reduction in RNA polymerase II occupancy occurs during nucleotide excision repair in the transcriptionally active loci like, RPB2 , PYK1 and RPL2B . These results collectively indicate that removal of RNAPII during DNA damage repair following UV irradiation is dependent on Rad26

Sensitivity of cells to transcription elongation inhibitors with or without UV treatment.

<p>Cells of exponentially growing cultures were appropriately diluted and spread on SC plates supplemented with MPA (A) or 6-AU (B) of indicated concentrations without UV irradiation. Similarly grown cells were spread on SC plates supplemented with MPA (C) or 6-AU (D) and subjected to UV doses as indicated. Growth was monitored after 72 h. For each strain, data represent the mean ±1 SD for four independent experiments.</p

Expression analyses of NER genes and <i>RPB2</i> gene.

<p>RT–PCR analysis was performed on total RNA isolated from WT and WTΔRad26 cells (<b>A</b>) following treatment without or with 100 J/m² UV radiation, using gene-specific primers, as described in Materials and Methods section; (<b>B</b>) of <i>RPB2</i> gene after 100 J/m² UV irradiation followed by repair incubation for different time periods. For each strain, data represent the mean ±1 SD for three independent experiments.</p

RNA polymerase II status during NER in different regions of the <i>RPB2</i> locus.

<p>ChIP analysis of RNA polymerase II status in different ORF regions of the <i>RPB2</i> locus as shown in (A), during NER. Cells were irradiated with 100 J/m² UV and incubated for different repair times as indicated. Chromatin was immunoprecipitated with 8WG16 antibody specific to RNA polymerase II, followed by quantitative PCR amplification using primers specific to ORF1 (B), ORF2 (C) and ORF3 (data not shown) of the <i>RPB2</i> locus in WT, H4 R45H, WTΔRad26 and H4R45HΔRad26 cells. The values given for ORF1 and ORF2 are calculated by normalizing the ChIP -PCR signal with the input PCR signal. The value for UV untreated cells was set as 1.0. For each strain, data represent the mean ±1 SD for four independent experiments. Corresponding ChIP-PCR, input-PCR and no antibody control gel pictures are given below each strain.</p

Domain organization of Rad26 protein.

<p>Bioinformatics based studies indicated the Rad26 protein to be composed of primarily three distinct domains. The N-terminal aspartate/glutamate-rich acidic domain; a SNF2 domain having the ATP-binding helicase sub-domain consisting of the ATP-binding pocket and a signature DEGH box; and the C-terminal helicase domain.</p