Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

SummaryPulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, a1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses.

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.

The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24) impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or ΔVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of ΔVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Clathrin assembles at bacterial adhesion sites and its phosphorylation is required for actin recruitment during bacterial infection.

Salmonella invades epithelial cells and survives within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time-points. Vacuole interactions with endoplasmic reticulum-derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper-replication within the host cytosol. On the other hand, SCV interactions with VAMP7-positive lysosome-like vesicles promote Salmonella-induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion.