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Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

IntroductionKlebsiella pneumoniae (Kp) is a common cause of hospital-acquired pneumonia. Although previous studies have suggested that evasion of phagocytic uptake is a virulence determinant of Kp, few studies have examined phagocytosis sensitivity in clinical Kp isolates.MethodsWe screened 19 clinical respiratory Kp isolates that were previously assessed for mucoviscosity for their sensitivity to macrophage phagocytic uptake, and evaluated phagocytosis as a functional correlate of in vivo Kp pathogenicity.ResultsThe respiratory Kp isolates displayed heterogeneity in the susceptibility to macrophage phagocytic uptake, with 14 out of 19 Kp isolates displaying relative phagocytosis-sensitivity compared to the reference Kp strain ATCC 43816, and 5 out of 19 Kp isolates displaying relative phagocytosis-resistance. Intratracheal infection with the non-mucoviscous phagocytosis-sensitive isolate S17 resulted in a significantly lower bacterial burden compared to infection with the mucoviscous phagocytosis-resistant isolate W42. In addition, infection with S17 was associated with a reduced inflammatory response, including reduced bronchoalveolar lavage fluid (BAL) polymorphonuclear (PMN) cell count, and reduced BAL TNF, IL-1β, and IL-12p40 levels. Importantly, host control of infection with the phagocytosis-sensitive S17 isolate was impaired in alveolar macrophage (AM)-depleted mice, whereas AM-depletion had no significant impact on host defense against infection with the phagocytosis-resistant W42 isolate.ConclusionAltogether, these findings show that phagocytosis is a primary determinant of pulmonary clearance of clinical Kp isolates.

Summary: Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Owing to cilia’s nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) from a defined number of cells. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and correlated it with the direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from patients with primary ciliary dyskinesia. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expedite the modeling of and therapeutic development for genetic and environmental ciliopathies.

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.