Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system

Polar Localization of Virulence-Related Esx-1 Secretion in Mycobacteria

The Esx-1 (type VII) secretion system is critical for virulence of both Mycobacterium tuberculosis and Mycobacterium marinum, and is highly conserved between the two species. Despite its importance, there has been no direct visualization of Esx-1 secretion until now. In M. marinum, we show that secretion of Mh3864, a novel Esx-1 substrate that remains partially cell wall–associated after translocation, occurred in polar regions, indicating that Esx-1 secretion takes place in these regions. Analysis of Esx-1 secretion in infected host cells suggested that Esx-1 activity is similarly localized in vivo. A core component of the Esx-1 apparatus, Mh3870, also localized to bacterial poles, showing a preference for new poles with active cell wall peptidoglycan (PGN) synthesis. This work demonstrates that the Esx-1 secretion machine localizes to, and is active at, the bacterial poles. Thus, virulence-related protein secretion is localized in mycobacteria, suggesting new potential therapeutic targets, which are urgently needed

A Mycobacterium ESX-1–Secreted Virulence Factor with Unique Requirements for Export

Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates

Host-Detrimental Role of Esx-1-Mediated Inflammasome Activation in Mycobacterial Infection

The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1β, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome

Atg5-Independent Sequestration of Ubiquitinated Mycobacteria

Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only ∼25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occured rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1–positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1–positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails

Resistance to phagocytosis in Streptococcus pyogenes

Phagocytosis of Streptococcus pyogenes is complement dependent. However, the cell wall-associated M protein, which exists in >100 different serotypes (M types), enables the bacteria to evade phagocytosis. To analyze the mechanisms by which M protein confers this ability we used, as model systems, two representative M proteins, the M22 and M5 proteins, which have properties typical for many M proteins of different M type

Vad är värdeskapande projektering?

Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p

En jämförelse av olika kommunikationstekniker och systemarkitekturiska lösningar vid uppbyggnad av nätverk i naturen.

Att konstruera ett tidtagningssystem för enduro i skogen leder till flera frågor som måste undersökas och besvaras. Vilken kommunikationsteknik ska användas och vilken systemarkitekturisk lösning passar bäst? Att dra kabel ute i skogen är ett bra sätt att lösa det kommunikationstekniska problemet, lösningen är säker och relativt billig. Visst påverkas priset av distansen mellan olika klienter ute i skogen, men alla positiva egenskaper hos tekniken gör denna kostnad värd sitt pris. De geografiska variationer som kan finnas är de som kan spela ut de övriga teknikerna i form av stabilitet. En snabb resultatförmedling kräver ett system som har en väl uppbyggd systemarkitektur. Vid tidtagning kommer data i form av tider skickas från klienterna direkt till huvudservern, ingen mellanlagring sker utan data lagras centralt vilket förespråkas i IRM. Dessutom kommer ingen redundans att accepteras, vilket även det förespråkas av IRM. Det finns även flera andra parametrar som väger för att välja IRM, vilket är det som rekommenderas utifrån den analys som gjorts i denna uppsats

En jämförelse av olika kommunikationstekniker och systemarkitekturiska lösningar vid uppbyggnad av nätverk i naturen.

Att konstruera ett tidtagningssystem för enduro i skogen leder till flera frågor som måste undersökas och besvaras. Vilken kommunikationsteknik ska användas och vilken systemarkitekturisk lösning passar bäst? Att dra kabel ute i skogen är ett bra sätt att lösa det kommunikationstekniska problemet, lösningen är säker och relativt billig. Visst påverkas priset av distansen mellan olika klienter ute i skogen, men alla positiva egenskaper hos tekniken gör denna kostnad värd sitt pris. De geografiska variationer som kan finnas är de som kan spela ut de övriga teknikerna i form av stabilitet. En snabb resultatförmedling kräver ett system som har en väl uppbyggd systemarkitektur. Vid tidtagning kommer data i form av tider skickas från klienterna direkt till huvudservern, ingen mellanlagring sker utan data lagras centralt vilket förespråkas i IRM. Dessutom kommer ingen redundans att accepteras, vilket även det förespråkas av IRM. Det finns även flera andra parametrar som väger för att välja IRM, vilket är det som rekommenderas utifrån den analys som gjorts i denna uppsats

Vad är värdeskapande projektering?

Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p