Dataset Information

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

ABSTRACT: Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.

SUBMITTER: Mell JC

PROVIDER: S-EPMC4849778 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

Mell Joshua Chang JC Viadas Cristina C Moleres Javier J Sinha Sunita S Fernández-Calvet Ariadna A Porsch Eric A EA St Geme Joseph W JW Nislow Corey C Redfield Rosemary J RJ Garmendia Junkal J

PLoS pathogens 20160428 4

Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic varia ...[more]

PMID: 27124727

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Project description:Recent technical advances have broadened our understanding of processes that govern mammalian cell migration in health and disease but many of the molecular and morphological alterations that precede and accompany movement of cells - in particular in three-dimensional (3D) environments - are still incompletely understood. In this manuscript, using high-resolution and time-lapse microscopy imaging approaches, we describe morphodynamic processes during rounded/amoeboid cell invasion and molecules associated with the cellular invasion structures. We used macrophages infected with the intracellular protozoan parasite Theileria annulata, which causes Tropical Theileriosis in susceptible ruminants such as domestic cattle. T. annulata transforms its host cell that, as a result, acquires many characteristics of human cancer cells including a markedly increased potential to migrate, disseminate and expand in the body of the host animal. Hence, virulence of the disease is associated with the capability of infected cells to disseminate inside the host. Using T. annulata-transformed macrophages as a model system, we described a novel mode of rounded/amoeboid macrophage migration. We show that filopodia-like membrane extensions at the leading edge lead the way and further evolve in blebbing membrane protrusions to promote progressive expansion of the matrix. Associated with focal invasion structures we detected ezrin, radixin, moesin-family proteins and their regulatory kinase MAP4K4. Furthermore, we linked Rho-kinase activity to contractile force generation, which is essential for infected cell motility. Thus, the motility mode of these parasite-transformed macrophages contrasts with those described so far in human macrophages such as the tunneling or mesenchymal modes, which require engulfment, compaction and ingestion of matrix or proteolytic matrix degradation, respectively. Together, our data reveal protrusion dynamics at the leading edge of invading cells in 3D at unprecedented temporal and spatial resolution and suggest a novel mode of rounded/amoeboid invasive cell motility that exploits actin-driven filopodia formation in combination with pressure-driven membrane blebs.

Project description:UnlabelledThe two-partner secretion (TPS) pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane pore-forming translocator protein. Previous work has demonstrated that the TpsA protein contains an N-terminal TPS domain that plays an important role in targeting the TpsB protein and is required for secretion. The nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins are homologous proteins that are prototype TpsA proteins and are secreted by the HMW1B and HMW2B TpsB proteins. In the present study, we sought to define the structural determinants of HMW1 interaction with HMW1B during the transport process and while anchored to the bacterial surface. Modeling of HMW1B revealed an N-terminal periplasmic region that contains two polypeptide transport-associated (POTRA) domains and a C-terminal membrane-localized region that forms a pore. Biochemical studies demonstrated that HMW1 engages HMW1B via interaction between the HMW1 TPS domain and the HMW1B periplasmic region, specifically, the predicted POTRA1 and POTRA2 domains. Subsequently, HMW1 is shuttled to the HMW1B pore, facilitated by the N-terminal region, the middle region, and the NPNG motif in the HMW1 TPS domain. Additional analysis revealed that the interaction between HMW1 and HMW1B is highly specific and is dependent upon the POTRA domains and the pore-forming domain of HMW1B. Further studies established that tethering of HMW1 to the surface-exposed region of HMW1B is dependent upon the external loops of HMW1B formed by residues 267 to 283 and residues 324 to 330. These observations may have broad relevance to proteins secreted by the TPS pathway.ImportanceSecretion of HMW1 involves a recognition event between the extended form of the HMW1 propiece and the HMW1B POTRA domains. Our results identify specific interactions between the HMW1 propiece and the periplasmic HMW1B POTRA domains. The results also suggest that the process of HMW1 translocation involves at least two discrete steps, including initial interaction between the HMW1 propiece and the HMW1B POTRA domains and then a separate translocation event. We have also discovered that the HMW1B pore itself appears to influence the translocation process. These observations extend our knowledge of the two-partner secretion system and may be broadly relevant to other proteins secreted by the TPS pathway.

Dataset Information

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

Publications

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

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