Host Defense Peptides LL-37 and Lactoferrin Trigger ET Release from Blood-Derived Circulating Monocytes.

Neutrophils are commonly regarded as the first line of immune response during infection or in tissue injury-induced inflammation. The rapid influx of these cells results in the release of host defense proteins (HDPs) or formation of neutrophil extracellular traps (NETs). As a second wave during inflammation or infection, circulating monocytes arrive at the site. Earlier studies showed that HDPs LL-37 and Lactoferrin (LTF) activate monocytes while neutrophil elastase facilitates the formation of extracellular traps (ETs) in monocytes. However, the knowledge about the impact of HDPs on monocytes remains sparse. In the present study, we investigated the effect of LL-37 and LTF on blood-derived CD14+ monocytes. Both HDPs triggered a significant release of TNFα, nucleosomes, and monocyte ETs. Microscopic analysis indicated that ET formation by LL-37 depends on storage-operated calcium entry (SOCE), mitogen-activated protein kinase (MAPK), and ERK1/2, whereas the LTF-mediated ET release is not affected by any of the here used inhibitors. Quantitative proteomics mass spectrometry analysis of the neutrophil granular content (NGC) revealed a high abundance of Lactoferrin. The stimulation of CD14+ monocytes with NGC resulted in a significant secretion of TNFα and nucleosomes, and the formation of monocyte ETs. The findings of this study provide new insight into the complex interaction of HDPs, neutrophils, and monocytes during inflammation.

YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis.

Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In Yersinia, one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

The Amino-Terminal Part of the Needle-Tip Translocator LcrV of Yersinia pseudotuberculosis Is Required for Early Targeting of YopH and In vivo Virulence.

Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal lcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.