Table 2.

Intracellular localization and fate of leptospires.

Leptospira spp.	Host cells	Main findings	Technical remarks	Ref
L. interrogans Icterohaemorrhagiae	Vero and J774A.1 cell lines	L. interrogans showed rapid internalization (20 min) that was lost after a few in vitro passages. Delayed or impaired internalization (60 min) of formalin-fixed and highly passaged strains. L. biflexa was extracellularly adherent. Slow internalization (60 min). Cytochalasin D: does not block internalization.	No gentamicin protection assay Double staining of extra- and intracellular bacteria	(18)
L. interrogans Lai L. biflexa Patoc	Vero and J774A.1 cell lines	Leptospires attached to host cells. Increased adherence in J774.1 compared to Vero cells. EM: “phagosome” and “lysosome” were observed. FCM: actin remodeling during infection. Colocalization of leptospires with the marker LAMP-1.	No gentamicin protection assay Important were controls missing	(19)
L. interrogans Lai (virulent), Pomona Luo (avirulent)	Human (THP-1 and primary Mφ) and murine (J77A.1, naive peritoneal cells and BMMs)	High adherence to all cell types. Murine cells: Leptospires were controlled (↓ CFU and viability). Membrane-associated bacteria showed a compromised shape. Increased colocalization with lysosomal markers over time. Human cells: Leptospires replicated (↑ CFU and viability). Cytosolic bacteria showed intact shapes. Replicative bacteria (CFU). Decreased colocalization with lysosomal markers over time.	No gentamicin protection assay Contradictory results compared to (21)/(22) Infection protocol ND	(20)
L. interrogans Lai, Luo L. biflexa Patoc	Vero and J774A.1 cell lines	L. interrogans [Lai (virulent) and Luo (avirulent)] but not L. biflexa adhered to cells (↑ adherence to Mφ). Lai and Luo observed inside of Vero and J774A.1 cells in the membranous compartment (EM).	No gentamicin protection assay	(23)
L. interrogans Lai	THP-1 and J774.1 cell lines	Leptospires triggered ROS production in both cell lines with no difference upon infection. Leptospires were intracellular (FCM) and associated with membranes (EM) in both cell lines.	Contradictory results compared to (20) Missing noninfected & nonstained controls	(21)
L. interrogans Manilae L. biflexa Patoc	C57BL/6 BMMs	Early - mid (1–6 hpi): intracellular leptospires colocalized with EEA-1 and LAMP-1. Saprophytic but not pathogenic leptospires showed a compromised shape. Infection with pathogenic strains led to delayed recruitment of cathepsin D and colocalization with LysoTracker. Late (24 hpi): only pathogenic strains were intracellularly membrane-associated (EM). Viable pathogenic bacteria were recovered in EMJH 24 and 48 hpi.	No gentamicin protection assay Double staining of extra- & intracellular bacteria	(24)
L. interrogans Manilae (low & high passage) lmb216/ligB mutants	C57BL/6 BMMs	The high-passage strain, ligB, and lmb216 (absent in L. biflexa) showed reduced adhesion and infection. Expression of LigB and Lmb216 in L. biflexa increased adhesion and infection of Mφ. Cytochalasin D partially reduced but did not block internalization.		(25)
L. interrogans Lai	THP-1 and J774.1 cell lines	Leptospires observed in phagosomes in both cell lines (EM).	Contradictory results compared to (20)	(22)
L. interrogans Pomona	Bovine PBMCs	More cells were infected with the virulent strain than the passage-attenuated strain. Production of IL-1β, TNF-α, and IL-10. Infection and colocalization with lysosomal markers were not affected by cytochalasin D.	No gentamicin protection assay	(26)

EM, electron microscopy; EFM, epi-fluorescence microscopy; FCM, fluorescence confocal microscopy; ROS, reactive oxygen species; EMJH, Ellinghausen-McCullough-Johnson-Harris culture media; pi, postinfection; Mφ, macrophages; EMC, extracellular matrix components; ND, nondescribed.

Host cells: THP-1, human monocyte cell line; J774.1, murine macrophage-like cell line; BMMs, bone marrow-derived macrophages; Vero, monkey kidney epithelial cells; PBMCs, peripheral blood mononuclear cells. In red, technical issues and/or studies that should be interpreted with caution because of a lack of controls and/or internally contradictory results. In green, techniques of interest.