The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here, we present a draft map of the human proteome using high resolution Fourier transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples including 17 adult tissues, 7 fetal tissues and 6 purified primary hematopoietic cells resulted in identification of proteins encoded by 17,294 genes accounting for ~84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream ORFs. This large human proteome catalog (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

SummaryBorrelia burgdorferi (Bb), the Lyme disease spirochaete, encodes a potential ferric uptake regulator (Fur) homologue, BosR (BB0647). Thus far, a role for BosR in Bb metabolism, gene regulation or pathogenesis has not been determined, largely due to the heretofore inability to inactivate bosR in low-passage, infectious Bb isolates. Herein, we report the generation of the first bosR-deficient mutant in a virulent strain of Bb. Whereas the bosR mutant persisted normally in ticks, the mutant was unable to infect mice, indicating that BosR is essential for Bb infection of a mammalian host. Moreover, transcriptional profiling of the bosR mutant showed that a number of genes were either positively or negatively influenced by BosR deficiency, suggesting that BosR may function both as a global repressor and activator in Bb. Strikingly, our study showed that BosR controls the expression of two major virulence-associated Bb lipoproteins, OspC and DbpA, likely via an influence on the alternative sigma factor, RpoS. This study thus not only has elucidated another key virulence gene of Bb, but also provides new insights into a previously unknown layer of gene regulation governing RpoS in Bb.

Calcium Dependent Protein Kinases are key effectors of calcium signaling in malaria parasite. PfCDPK1 is critical for asexual development of Plasmodium falciparum, but its precise function and substrates remain largely unknown. Using a conditional knockdown strategy, we here establish that this kinase is critical for the invasion of host erythrocytes. Furthermore, using a multidisciplinary approach involving comparative phosphoproteomics we gain insights into the underlying molecular mechanisms. We identify substrates of PfCDPK1, which includes proteins of Inner Membrane Complex and glideosome-actomyosin motor assembly. Interestingly, PfCDPK1 phosphorylates PfPKA regulatory subunit (PfPKA-R) and regulates PfPKA activity in the parasite, which may be relevant for the process of invasion. This study delineates the signaling network of PfCDPK1 and sheds light on mechanisms via which it regulates invasion.

Borrelia burgdorferi bb0323 encodes an immunogenic protein in mammalian hosts including humans. An analysis of bb0323 expression in vivo showed variable transcription throughout the spirochete infection cycle, with elevated expression during tick-mouse transmission. Deletion of bb0323 in infectious B. burgdorferi did not affect microbial survival in vitro, despite significant alterations in growth kinetics and cell morphology. bb0323 mutants were unable to infect either mice or ticks, and were quickly eliminated from immunocompetent and immunodeficient hosts and the vector, within the first few days of inoculation. Chromosomal complementation of the mutant with native bb0323 and phenotypic analysis in vivo indicated the significant restoration of spirochete virulence and persistence throughout the mouse-tick infection cycle. BB0323 may serve an indispensable physiological function that is more pronounced during microbial persistence and transitions between the host and the vector in vivo. Strategies to interfere with BB0323 function may interrupt the infectious cycle of spirochetes.

Borrelia burgdorferi, the pathogen of Lyme disease, cycles in nature through Ixodes ticks and mammalian hosts. At least five Complement Regulator-Acquiring Surface Proteins (BbCRASPs) are produced by B. burgdorferi, which are thought to assist spirochetes in host immune evasion. Recent studies established that BbCRASP-2 is preferentially expressed in mammals, and elicits robust antibody response in infected hosts, including humans. We show that BbCRASP-2 is ubiquitously expressed in diverse murine tissues, but not in ticks, reinforcing a role of BbCRASP-2 in conferring B. burgdorferi defense against persistent host immune threats, such as complement. BbCRASP-2 immunization, however, fails to protect mice from B. burgdorferi infection and does not modify disease, as reflected by the development of arthritis. An infectious BbCRASP-2 mutant was generated, therefore, to examine the precise role of the gene product in spirochete infectivity. Similar to wild type B. burgdorferi, BbCRASP-2 mutants remain insensitive to complement-mediated killing in vitro, retain full murine infectivity and induce arthritis. Quantitative RT-PCR assessment indicates that survivability of BbCRASP-2-deficient B. burgdorferi is not due to altered expression of other BbCRASPs. Together, these results suggest that the function of a selectively expressed B. burgdorferi gene, BbCRASP-2, is not essential for complement resistance or infectivity in the murine host.

Borrelia burgdorferi, the pathogen of Lyme borreliosis, persists in nature through a tick-rodent transmission cycle. A selective assessment of the microbial transcriptome, limited to gene encoding putative membrane proteins, reveals that bba52 transcription in vivo is strictly confined to the vector-specific portion of microbial life cycle with highest expression levels in feeding ticks and swift downregulation in mice. bba52 deletion did not affect murine disease as assessed by the genesis of arthritis and carditis or long-term pathogen persistence in mice or ticks. However, bba52 deficiency did impair microbial transitions between hosts and vector, defects that could be fully rescued when bba52 expression was genetically restored to the original genomic locus. These studies establish that BBA52 facilitates vector-host transitions by the pathogen and as such, is a potential antigenic target for interference with B. burgdorferi transmission from ticks to mammalian hosts.

Borrelia burgdorferi lipoprotein Lp6.6 is a differentially produced spirochete antigen. An assessment of lp6.6 expression covering representative stages of the infectious cycle of spirochetes demonstrates that the gene is solely expressed during pathogen persistence in ticks. Deletion of lp6.6 in infectious B. burgdorferi did not influence in vitro growth, or its ability to persist and induce inflammation in mice, migrate to larval or nymphal ticks or survive through the larval-nymphal molt. However, Lp6.6-deficient spirochetes displayed significant impairment in their ability to transmit from infected ticks to naïve mice, which was restored upon genetic complementation of the mutant with a wild-type copy of lp6.6, establishing that Lp6.6 plays a role in pathogen transmission from ticks to mammals. Lp6.6 is a subsurface, yet highly abundant, outer membrane antigen. Two-dimensional blue native/SDS-PAGE coupled with liquid chromatography-mass spectrometry (LC-MS/MS) analysis and protein cross-linking studies independently shows that Lp6.6 exists in multiple protein complexes in the outer membrane. We speculate that the function of Lp6.6 is connected to the physiological processes of these membrane complexes. Further characterization of differentially produced membrane antigens and associated protein complexes will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.

BackgroundThe maintenance of Borrelia burgdorferi in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in B. burgdorferi plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of B. burgdorferi, has been lacking.ResultsHerein, we examined the expression of rpoS and key lipoprotein genes regulated by RpoS, including ospC, ospA, and dbpA, throughout the entire tick-mammal infectious cycle of B. burgdorferi. Our data revealed that transcription of rpoS, ospC, and dbpA is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of rpoS and dbpA in B. burgdorferi within mouse tissues following borrelial dissemination. However, dbpA transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the dbpBA operon. Our results also provide further evidence for the downregulation of ospA expression during mammalian infection, and the repression of ospC at later phases of mammalian infection by B. burgdorferi.ConclusionOur study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of B. burgdorferi to its mammalian host, and is sustained during mammalian infection.