TABLE 1

Reduced macrophage infectivity of LeishIF4E-3(+/−) mutanta

ParameterCell lineMeanSDP valued
% infected cellsb
    24 hWT84.609.80***
Cas9/T780.398.43**
4E-3(+/−)49.737.75
Addback84.419.89**
    4 hWT95.795.43**
Cas9/T787.506.66**
4E-3(+/−)39.3210.15
Addback96.304.79**
No. of parasites per infected cellc
    4 hWT4.681.50**
Cas9/T73.951.06**
4E-3(+/−)1.580.40
Addback4.531.16**
    24 hWT12.602.82***
Cas9/T711.901.07***
4E-3(+/−)2.140.28
Addback13.964.39***
  • a The ability of the LeishIF4E-3(+/−) mutant to infect the macrophage RAW 264.7 cells was quantified by counting the number of infected macrophages. Control infections included wild-type parasites, the Cas9/T7-expressing cells, and the LeishIF4E-3 addback cells, in which expression of LeishIF4E-3 was recovered.

  • b Infectivity of each cell line was monitored by confocal microscopy as described in the legend to Fig. 6, at 24 and 4 h postinfection.

  • c Parasite loads in infected macrophages were also determined, by counting the number of parasites per infected macrophage. The cells were counted using a cell counting plugin of Image J. A total of 100 macrophages were counted three times for each parasite cell line.

  • d The statistical analysis for percentage of infected cells and average parasite per cell, along with standard deviation values (SD), was performed by Kruskal-Wallis test using GraphPad Prism. The number of infected macrophages and average number of parasite counts per infected macrophage were the two parameters that were compared between LeishIF4E-3(+/−) deletion mutants and each of the control lines: wild type, transgenic cells expressing Cas9/T7, and LeishIF4E-3 addback cells. ***, P < 0.001; **, P < 0.01. The statistical difference between the control lines was nonsignificant.